GAS57 mutant antigens and GAS57 antibodies

ABSTRACT

The invention provides mutants of GAS57 (Spy0416) which are unable to cleave IL-8 and similar substrates but which still maintain the ability to induce protection against  S. pyogenes . The invention also provides antibodies which specifically bind to GAS57 and which inhibit its ability to cleave IL-8 and similar substrates. The mutants are useful, inter alia, in vaccine compositions to induce protection against  S. pyogenes . The antibodies are useful, e.g., as therapeutics for treating  S. pyogenes  infections.

This application is a division of Ser. No. 13/607,990, filed on Sep. 10,2011 and issued as U.S. Pat. No. 8,399,651, which is a division of Ser.No. 12/676,192, filed on Jan. 5, 2011 and issued as U.S. Pat. No.8,287,885, which is a national stage application of PCT/IB2008/003078filed on Sep. 12, 2008, which claims priority to Ser. No. 60/971,637filed on Sep. 12, 2007.

This application incorporates by reference the contents of a 756 kb textfile created on Sep. 10, 2012 and named “PAT052285_sequencelisting.txt,”which is the sequence listing for this application.

FIELD OF THE INVENTION

This invention is in the fields of immunology and vaccinology. Inparticular, it relates to antigens derived from Streptococcus pyogenesand their use in immunization.

BACKGROUND OF THE INVENTION

S. pyogenes (Group A Streptococcus; GAS) antigen GAS57, expressed asrecombinant protein and purified from E. coli, induces protectiveactivity against a lethal challenge with S. pyogenes in mice. However,GAS57 is a protease which cleaves and inactivates human chemokines suchas interleukin-8 (IL-8) (Edwards et al., J Infectious Diseases 192,783-90, 2005; Hidalgo-Grass et al., EMBO J 25, 4628-37, 2006). Thisproperty of GAS57 may hamper its use in a vaccine composition, due topossible side effects. Thus, there is a need in the art for GAS57antigens which are unable to cleave human chemokines but which stillmaintain the ability to induce protection against S. pyogenes. There isalso a need in the art for antibodies which specifically bind to GAS57antigens and which impair the ability of GAS57 to cleave IL-8 and othersubstrates.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Photomicrograph of an SDS-polyacrylamide gel demonstratingcleavage of IL-8 by wild-type GAS57.

FIGS. 2A-B. BLAST alignment of wild-type GAS57 (query, SEQ ID NO:1) vs aC5a peptidase serine protease (Sbjct, SEQ ID NO:9). FIG. 2A, alignmentof amino acids 1-650 of SEQ ID NO:1 and amino acids 1-545 of SEQ IDNO:9. FIG. 2B, alignment of amino acids 651-1278 of SEQ ID NO:1 andamino acids 546-1181 of SEQ ID NO:9.

FIG. 3. Photomicrograph of SDS-polyacrylamide gels demonstrating thatGAS57 point mutant D151A has lost the ability to cleave IL-8.

FIG. 4. Graph showing the results of an ELISA assay demonstrating thatGAS57 point mutant D151A has lost the ability to cleave IL-8.

FIGS. 5A-B. Photomicrographs of SDS-polyacrylamide gels demonstratingthat GAS57 single mutants D151A and S617A and the double mutantD151A+S617A have lost GAS57 proteolytic activity.

FIG. 6. Graph showing the results of an ELISA assay demonstrating thatsingle mutants D151A and S617A and GAS57 double mutant D151A+S617A havelost GAS57 proteolytic activity.

FIG. 7. Photomicrograph of an SDS-polyacrylamide gel demonstrating thatwild-type GAS57 is post-translationally modified into two polypeptidefragments of 150.5 kDa and 23.4 kDa.

FIG. 8. Photomicrograph of an SDS-polyacrylamide gel demonstrating thatGAS57 mutants D151A, S617A, and D151A+S617A are not post-translationallymodified into two polypeptide fragments of 150.5 kDa and 23.4 kDacompared to wild-type (black arrows). A major band of 174 kDacorresponding to unprocessed protein is instead present in the lanescorresponding to inactive mutant strains (grey arrow).

FIGS. 9A-B. ELISA assay results demonstrating dose-dependent inhibitionof GAS57-mediated IL-8 cleavage by polyclonal antisera against GAS57 intwo different experimental conditions. FIG. 9A, 8 hour incubation, 0.1μg/ml of GAS57. FIG. 9B, 24 hour incubation, 0.05 μg/ml of GAS57.

FIG. 10A-GG. Alignments of GAS57 antigens from different strains/Mtypes. The catalytic triad (D, H, S) is in bold black characters. FIG.10A, amino acids 1-50 (amino acid numbers at the top of each of FIGS.10A-GG refers to the amino acid sequence of gas57M1_SF370, SEQ ID NO:1);FIG. 10B, amino acids 51-100; FIG. 10C, amino acids 101-150; FIG. 10D,amino acids 151-200; FIG. 10E, amino acids 201-250; FIG. 10F, aminoacids 251-300; FIG. 10G, amino acids 301-350; FIG. 1H, amino acids351-400, FIG. 10I, amino acids 401-450; FIG. 10J, amino acids 451-500;FIG. 10K, amino acids 501-550; FIG. 10L, amino acids 551-600; FIG. 10M,amino acids 601-650; FIG. 10N, amino acids 651-700; FIG. 10O, aminoacids 701-750; FIG. 10P, amino acids 751-800; FIG. 10Q, amino acids801-850; FIG. 10R, amino acids 851-900; FIG. 10S, amino acids 901-950;FIG. 10T, amino acids 951-1000; FIG. 10U, amino acids 1001-1050; FIG.10V, amino acids 1051-1100; FIG. 10W, amino acids 1101-1150; FIG. 10X,amino acids 1151-1200; FIG. 10Y, amino acids 1201-1250; FIG. 10Z, aminoacids 1251-1300; FIG. 10AA, amino acids 1301-1350; FIG. 10BB, aminoacids 1351-1400; FIG. 10CC, amino acids 1401-1450; FIG. 10DD, aminoacids 1451-1500; FIG. 10EE, amino acids 1501-1550; FIG. 10FF, aminoacids 1551-1600; FIG. 10GG, amino acids 1601-1650. gas57M1_SF370, SEQ IDNO:1; gas57M1_(—)31075, SEQ ID NO:10; gas57M1_(—)31237, SEQ ID NO:11;gas57M1_(—)3348, SEQ ID NO:12; gas57M2_(—)34585, SEQ ID NO:13;gas57M3,1_(—)21398, SEQ ID NO:14; gas57M44-61_(—)20839, SEQ ID NO:15;gas57M6,31_(—)20022, SEQ ID NO:16; gas57M11_(—)20648, SEQ ID NO:17;gas57M23_(—)2071, SEQ ID NO:18; gas57M18,3_(—)40128, SEQ ID NO:19;gas47M4_(—)10092, SEQ ID NO:20; gas57M4_(—)30968, SEQ ID NO:21;gas57M6,31_(—)22692, SEQ ID NO:22; gas57M68,5_(—)22814, SEQ ID NO:23;gas57M68_(—)23623, SEQ ID NO:24; gas57M2_(—)10064, SEQ ID NO:25;gas57M2_(—)10065, SEQ ID NO:26; gas57M77_(—)10251, SEQ ID NO:27;gas57M77_(—)10527, SEQ ID NO:28; gas57M77_(—)20696, SEQ ID NO:29;gas57M89_(—)21915, SEQ ID NO:30; gas57M89_(—)23717, SEQ ID NO:31;gas57M94_(—)10134, SEQ ID NO:32; gas57M28_(—)10164, SEQ ID NO:33;gas57M28_(—)10218, SEQ ID NO:34; gas57M29_(—)10266, SEQ ID NO:35;gas57M28_(—)10299, SEQ ID NO:36; gas57M28_(—)30176, SEQ ID NO:37;gas57M28_(—)30574, SEQ ID NO:38; gas57M6,9_(—)21802, SEQ ID NO:39;gas57M75_(—)20671, SEQ ID NO:40; gas57M75_(—)30603, SEQ ID NO:41;gas57M75_(—)30207, SEQ ID NO:42; gas57M22_(—)20641, SEQ ID NO:43;gas57M22_(—)23465, SEQ ID NO:44; gas57M3,1_(—)30610, SEQ ID NO:45;gas57M3,1_(—)40603, SEQ ID NO:46; gas57M3,28_(—)24214, SEQ ID NO:47;gas57M3,34_(—)10307, SEQ ID NO:48; gas57M4_(—)40427, SEQ ID NO:49;gas57M3_(—)2721, SEQ ID NO:50; gas57M12_(—)10296, SEQ ID NO:51;gas57M12_(—)10035, SEQ ID NO:52; gas57M12_(—)20069, SEQ ID NO:53;gas57M12_(—)22432, SEQ ID NO:54; gas57M4_(—)40499, SEQ ID NO:55; andgas57M6,1_(—)21259, SEQ ID NO:56; gas57M75_(—)20671, SEQ ID NO:80.

FIG. 11. Alignment of human chemokines. GAS57 cleaves CXCL8 (IL-8) (SEQID NO:81) between the two bolded and underlined amino acids. CXCL4, SEQID NO:57; CXCL7/NAP-2, SEQ ID NO:58; CXCL1/GROα, SEQ ID NO:59;CXCL2/GROβ, SEQ ID NO:60; CXCL3/GROγ, SEQ ID NO:61; CXCL6/GCP-2, SEQ IDNO:62; CXCL12/SDF-1α, SEQ ID NO:63; CXCL12/SDF-1γ, SEQ ID NO:64;CXCL12/SDF-1β, SEQ ID NO:65; CXCL9/MIG, SEQ ID NO:66; CXCL10/IP10, SEQID NO:67; and CXCL11, SEQ ID NO:68.

FIG. 12. Photomicrograph of SDS-polyacrylamide gels demonstratingcleavage of CXC chemokines by GAS57.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides mutants of Spy0416 or GAS57 (referred to hereinas “GAS57 mutant antigens,” “GAS57 mutants,” “mutant GAS57 antigens”)which are unable to cleave human chemokines like IL-8 but which stillmaintain the ability to induce protection against S. pyogenes. GAS57mutants of the invention are useful in vaccine compositions, to induceprotection against S. pyogenes. The invention also provides antibodieswhich specifically bind to wild-type GAS57 and which inhibit the abilityof GAS57 to cleave IL-8 and similar substrates. It is envisaged that theantibodies will be useful as therapeutics for the prevention and/ortreatment of S. pyogenes infections.

Mutant GAS57 Antigens

“GAS57” is also referred to as ‘Spy0416’ (M1), ‘SpyM3_(—)0298’ (M3),‘SpyM18_(—)0464’ (M18) and ‘prtS.’ GAS57 has also been identified as aputative cell envelope proteinase. See WO 02/34771 and US 2006/0258849.There are 49 GAS57 sequences from 17 different M types (1, 2, 3, 4, 6,11, 12, 18, 22, 23, 28, 44/61, 68, 75, 77, 89, 94); according to theCenters for Disease Control, the 17 different M types account for over95% of pharyngitis cases and about 68% of the invasive GAS isolates inthe United States. The amino acid sequences of wild-type GAS57 antigensare set forth in the sequence listing as SEQ ID NOS:1, 10-56, and 80.Wild-type GAS57 contains two non-covalently associated peptides (seeExample 5 and FIG. 7).

Mutant GAS57 antigens according to the invention have a proteolyticactivity against interleukin 8 (IL-8) which is reduced by at least 50%(e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%)relative to wild-type GAS57 as detected by either SDS-PAGE or ELISA (seeExamples 2 and 3), but are immunogenic, e.g., they confer protectionagainst GAS lethal challenge in a mouse model (Example 4). Preferably, amutant GAS57 of the invention also does not cleave other humancytokines, such as CXCL1/GROα (e.g., SEQ ID NO:59), CXCL2/GROβ (e.g.,SEQ ID NO:60), CXCL3/GROγ (e.g., SEQ ID NO:61), CXCL4 (e.g., SEQ IDNO:57), CXCL12/SDF-1α (e.g., SEQ ID NO:63), CXCL12/SDF-1β (e.g., SEQ IDNO:65), CXCL12/SDF-1γ (e.g., SEQ ID NO:64), CXCL5/ENA78 (e.g, SEQ IDNO:82), CXCL6/GCP-2 (e.g., SEQ ID NO:62), CXCL7/NAP-2 (e.g., SEQ IDNO:58), CXCL9/MIG (e.g., SEQ ID NO:66), CXCL10/IP10 (e.g., SEQ IDNO:67), CXCL11 (e.g., SEQ ID NO:68), CXCL13 (e.g., SEQ ID NO:83), CXCL14(e.g., SEQ ID NO:84), and CXCL16 (e.g., SEQ ID NO:85). Unexpectedly,GAS57 mutants of the invention are single polypeptides, in contrast towild-type GAS57, which undergoes post-translational processing(maturation) to form two non-covalently associated peptides (Examples 5and 6). The ability to obtain such antigens in the form of a singlepeptide facilitates the production of the recombinant protein forvaccine purposes.

GAS57 mutants of the invention include those with at an amino acidalteration (i.e., a substitution, deletion, or insertion) at one or moreof amino acids D151, H279, or S617, numbered according to the wild-typeGAS57 sequence shown in SEQ ID NO:1 (see FIG. 10).

GAS57 mutants of the invention include single, double, or triple aminoacid alterations (“single mutants,” “double mutants,” “triple mutants”)at positions D151, H279, and/or S617. Thus, GAS57 mutants can comprisethe following:

-   -   i. D151A (SEQ ID NO:2), D151R, 151N, D151C, D151Q, D151E, D151G,        D151H, D151I, D151L, D151K, D151M, D151F, D151P, D151S, D151T,        D151W, D151Y, or D151V;    -   ii. H279A, H279R, H279N, H279D, H279C, H279Q, H279E, H279G,        H279I, H279L, H279K, H279M, H279F, H279P, H279S, H279T, H279W,        H279Y, or H279V;    -   iii. S617A (SEQ ID NO:3), S617R, S617N, S617D, S617C, S617Q,        S617E, S617G, S617H, S617I, S617L, S617K, S617M, S617F, S617P,        S617T, S617W, S617Y, or S617V;    -   iv. ΔD151; or ΔH279; or ΔS617; and    -   v. combinations thereof, such as D151A+S617A (SEQ ID NO:4).

GAS57 mutant antigens of the invention also include fusion polypeptideswhich comprise a GAS57 mutant antigen as disclosed above and another GASantigen. GAS antigens are disclosed, e.g., in WO 02/34771 and include,but are not limited to, GAS25 (Spy0167; gi13621460) GAS39 (Spy0266;gi13621542), GAS40 (Spy0269; gi13621545), GAS42 (Spy0287; gi13621559),GAS45 (M5005_Spy0249; gi71910063), GAS58 (Spy0430; gi13621663), GAS84(SPy1274; 13622398), GAS95 (SPy1733; 13622787), GAS117 (Spy0448;gi13621679), GAS130 (Spy0591; gi13621794), GAS137 (Spy0652; gi13621842),GAS159 (Spy1105; gi13622244), GAS193 (Spy2025; gi3623029), GAS202(Spy1309; gi13622431), GAS217 (Spy0925, gi1362208), GAS236 (Spy1126;gi13622264), GAS253 (Spy1524; gi13622611), GAS277 (Spy1939; gi13622962),GAS294 (Spy1173; gi13622306), GAS309 (Spy0124; gi13621426), GAS366(Spy1525; gi13622612), GAS372 (Spy1625; gi13622698), GAS384 (Spy1874;gi13622908), GAS389 (Spy1981; gi13622996), GAS504 (Spy1751; gi13622806),GAS509 (Spy1618; gi13622692), GAS290 (SPy1959; gi13622978), GAS511(Spy1743; gi13622798), GAS527 (Spy1204; gi3622332), GAS529 (Spy1280;gi3622403), and GAS533 (Spy1877; gi13622912). GAS antigens also include,GAS68 (Spy0163; gi13621456), GAS84 (Spy1274; gi13622398), GAS88(Spy1361; gi13622470), GAS89 (Spy1390; gi13622493), GAS98 (Spy1882;gi13622916), GAS99 (Spy1979; gi13622993), GAS102 (Spy2016, gi13623025),GAS146 (Spy0763; gi13621942), GAS195 (Spy2043; gi13623043), GAS561(Spy1134; gi13622269), GAS179 (Spy1718, gi13622773) and GAS681 (spy1152;gi1362228).

The invention also includes equivalents of GAS57 mutants which aresingle polypeptides, which do not cleave IL-8 as determined by SDS-PAGEor ELISA, which are immunogenic, and which confer protection against GASlethal challenge in a mouse model. Such equivalents may include mutantGAS57 antigens with amino acid deletions, insertions, and/orsubstitutions at positions other than D151, H279, or S617, includingdeletions of up to about 40 amino acids at the N or C terminus (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,or 40 amino acids). Such equivalents thus include GAS57 mutants havingdeletions, insertions, and/or substitutions at positions other thanD151, H279, or S617 in addition to having an amino acid alteration atone or more of amino acids D151, H279 or S617 and S617, as describedabove.

Nucleic Acid Molecules

The invention includes nucleic acid molecules which encode mutant GAS57antigens.

The invention also includes nucleic acid molecules comprising nucleotidesequences having at least 50% sequence identity to such molecules.Depending on the particular sequence, the degree of sequence identity ispreferably greater than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). Identity betweennucleotide sequences is preferably determined by the Smith-Watermanhomology search algorithm as implemented in the MPSRCH program (OxfordMolecular), using an affine gap search with parameters gap openpenalty=12 and gap extension penalty=1.

The invention also provides nucleic acid molecules which can hybridizeto these molecules. Hybridization reactions can be performed underconditions of different “stringency.” Conditions which increasestringency of a hybridization reaction are widely known and published inthe art. See, e.g., page 7.52 of Sambrook et al., Molecular Cloning: ALaboratory Manual, 1989. Examples of relevant conditions include (inorder of increasing stringency): incubation temperatures of 25° C., 37°C., 50° C., 55° C., and 68° C.; buffer concentrations of 10×SSC, 6×SSC,1×SSC, and 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer)and their equivalents using other buffer systems; formamideconcentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutesto 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2,or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, orde-ionized water. Hybridization techniques and their optimization arewell known in the art. See, e.g., Sambrook, 1989; Ausubel et al., eds.,Short Protocols in Molecular Biology, 4th ed., 1999; U.S. Pat. No.5,707,829; Ausubel et al., eds., Current Protocols in Molecular Biology,Supplement 30, 1987.

In some embodiments, nucleic acid molecules of the invention hybridizeto a target under low stringency conditions; in other embodiments,nucleic acid molecules of the invention hybridize under intermediatestringency conditions; in preferred embodiments, nucleic acid moleculesof the invention hybridize under high stringency conditions. An exampleof a low stringency hybridization condition is 50° C. and 10×SSC. Anexample of an intermediate stringency hybridization condition is 55° C.and 1×SSC. An example of a high stringency hybridization condition is68° C. and 0.1×SSC.

Production of Mutant GAS57 Antigens

Recombinant Production

The redundancy of the genetic code is well-known. Thus, any nucleic acidmolecule (polynucleotide) which encodes wild-type GAS57 protein or aGAS57 mutant protein of the invention can be used to produce thatprotein recombinantly. Examples of nucleotide sequences which encodewild-type GAS57, GAS57 mutant D151A, GAS57 mutant S617A, and GAS mutantD151A+S617A are provided in SEQ ID NOS:5, 6, 7, and 8, respectively.Nucleic acid molecules encoding wild-type GAS57 also can be isolatedfrom the appropriate S. pyogenes bacterium using standard nucleic acidpurification techniques or can be synthesized using an amplificationtechnique, such as the polymerase chain reaction (PCR), or by using anautomatic synthesizer. See Caruthers et al., Nucl. Acids Res. Symp. Ser.215 223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225 232, 1980;Hunkapiller et al, Nature 310, 105-11, 1984; Grantham et al., NucleicAcids Res. 9, r43-r74, 1981.

cDNA molecules can be made with standard molecular biology techniques,using mRNA as a template. cDNA molecules can thereafter be replicatedusing molecular biology techniques well known in the art. Anamplification technique, such as PCR, can be used to obtain additionalcopies of polynucleotides of the invention, using either genomic DNA orcDNA as a template.

If desired, polynucleotides can be engineered using methods generallyknown in the art to alter antigen-encoding sequences for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing, and/or expression of the polypeptide or mRNAproduct. DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides can be used to engineerthe nucleotide sequences. For example, site directed mutagenesis can beused to insert new restriction sites, alter glycosylation patterns,change codon preference, produce splice variants, introduce mutations,and so forth.

Sequence modifications, such as the addition of a purification tagsequence or codon optimization, can be used to facilitate expression.For example, the N-terminal leader sequence may be replaced with asequence encoding for a tag protein such as polyhistidine (“HIS”) orglutathione S-transferase (“GST”). Such tag proteins may be used tofacilitate purification, detection, and stability of the expressedprotein. Codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producean RNA transcript having desirable properties, such as a half life whichis longer than that of a transcript generated from the naturallyoccurring sequence. These methods are well known in the art and arefurther described in WO05/032582.

Expression Vectors

A nucleic acid molecule which encodes a mutant GAS57 antigen can beinserted into an expression vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing coding sequences and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination.

Host Cells

Host cells for producing mutant GAS57 antigens can be prokaryotic oreukaryotic. E. coli is a preferred host cell, but other suitable hostsinclude Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis,Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisserialactamica, Neisseria cinerea, Mycobacteria (e.g., M. tuberculosis),yeasts, baculovirus, mammalian cells, etc.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedpolypeptide in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation. Posttranslational processing which cleaves a “prepro” form of thepolypeptide also can be used to facilitate correct insertion, foldingand/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post translationalactivities are available from the American Type Culture Collection(ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can bechosen to ensure the correct modification and processing of a foreignprotein. See WO 01/98340.

Expression constructs can be introduced into host cells usingwell-established techniques which include, but are not limited to,transferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun” methods, and DEAE-or calcium phosphate-mediated transfection.

Host cells transformed with expression vectors can be cultured underconditions suitable for the expression and recovery of the protein fromcell culture. The protein produced by a transformed cell can be secretedor contained intracellularly depending on the nucleotide sequence and/orthe expression vector used. Those of skill in the art understand thatexpression vectors can be designed to contain signal sequences whichdirect secretion of soluble antigens through a prokaryotic or eukaryoticcell membrane.

Purification

Signal export sequences can be included in a recombinantly producedmutant GAS57 antigen so that the antigen can be purified from cellculture medium using known methods. Alternatively, recombinantlyproduced mutant GAS57 antigens of the invention can be isolated fromengineered host cells and separated from other components in the cell,such as proteins, carbohydrates, or lipids, using methods well-known inthe art. Such methods include, but are not limited to, size exclusionchromatography, ammonium sulfate fractionation, ion exchangechromatography, affinity chromatography, and preparative gelelectrophoresis. A preparation of purified mutant GAS57 antigens is atleast 80% pure; preferably, the preparations are 90%, 95%, or 99% pure.Purity of the preparations can be assessed by any means known in theart, such as SDS-polyacrylamide gel electrophoresis. Where appropriate,mutant GAS57 antigens can be solubilized, for example, with urea.

Chemical Synthesis

Mutant GAS57 antigens can be synthesized, for example, using solid phasetechniques. See, e.g., Merrifield, J. Am. Chem. Soc. 85, 2149 54, 1963;Roberge et al., Science 269, 202 04, 1995. Protein synthesis can beperformed using manual techniques or by automation. Automated synthesiscan be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Optionally, fragments of a mutant GAS57antigen can be separately synthesized and combined using chemicalmethods to produce a full-length molecule.

GAS57 Antibodies

The invention also provides antibodies which specifically bind towild-type GAS57 and which substantially reduce or eliminate the abilityof GAS57 to cleave IL-8. Some antibodies of the invention alsospecifically bind to mutant GAS57 as described above. Preferredantibodies also reduce or eliminate the ability of GAS57 to cleave othersubstrates such as homologs of IL-8 (e.g., CXCL1/GROα, CXCL2/GROβ,CXCL3/GROγ, CXCL4, CXCL12/SDF-1α, CXCL12/SDF-1β, CXCL12/SDF-1γ,CXCL5/ENA 78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL9/MIG, CXCL10/IP10, CXCL11,CXCL13, CXCL14, and CXCL16). An antibody “specifically binds” towild-type or mutant GAS57 if it provides a detection signal at least 5-,10-, or 20-fold higher than a detection signal provided with non-GAS57proteins when used in an immunochemical assay. Preferably, antibodiesthat specifically bind to wild-type or mutant GAS57 do not detectnon-GAS57 proteins in immunochemical assays and can immunoprecipitatewild-type GAS57 from solution. Antibodies according to the invention mayreduce the proteolytic activity of GAS57 against interleukin 8 (IL-8) byat least 50% (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98,99, or 100%) relative to wild-type GAS57 as detected by either SDS-PAGEor ELISA.

In preferred embodiments, antibodies of the invention block theprogression of necrotic lesions in animals immunized with wild-type ormutant GAS57 recombinant antigen and challenged with GAS.

“Antibody” as used herein includes intact immunoglobulin molecules, aswell as fragments thereof which specifically bind to wild-type GAS57and, in some cases, to a GAS57 mutant antigen. These include hybrid(chimeric) antibody molecules (e.g., Winter et al., Nature 349, 293-99,1991; U.S. Pat. No. 4,816,567); F(ab′)₂ and F(ab) fragments and F_(v)molecules; non-covalent heterodimers (e.g., Inbar et al., Proc. Natl.Acad. Sci. U.S.A. 69, 2659-62, 1972; Ehrlich et al., Biochem 19,4091-96, 1980); single-chain Fv molecules (sFv) (e.g., Huston et al.,Proc. Natl. Acad. Sci. U.S.A. 85, 5897-83, 1988); dimeric and trimericantibody fragment constructs; minibodies (e.g., Pack et al., Biochem 31,1579-84, 1992; Cumber et al., J. Immunology 149B, 120-26, 1992);humanized antibody molecules (e.g., Riechmann et al., Nature 332,323-27, 1988; Verhoeyan et al., Science 239, 1534-36, 1988; and U.K.Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and anyfunctional fragments obtained from such molecules, as well as antibodiesobtained through non-conventional processes such as phage display.

Generation of GAS57 Antibodies

A mutant or wild-type GAS57 antigen can be used to immunize a mammal,such as a mouse, rat, rabbit, guinea pig, monkey, or human, to producepolyclonal antibodies. If desired, a GAS57 antigen can be conjugated toa carrier protein, such as bovine serum albumin, thyroglobulin, andkeyhole limpet hemocyanin. Depending on the host species, variousadjuvants can be used to increase the immunological response. Suchadjuvants include, but are not limited to, Freund's adjuvant, mineralgels (e.g., aluminum hydroxide), and surface active substances (e.g.lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used inhumans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum areespecially useful.

Monoclonal antibodies which specifically bind to a wild-type or mutantGAS57 antigen can be prepared using any technique which provides for theproduction of antibody molecules by continuous cell lines in culture.These techniques include, but are not limited to, the hybridomatechnique, the human B cell hybridoma technique, and the EBV hybridomatechnique (Kohler et al., Nature 256, 495-97, 1985; Kozbor et al., J.Immunol. Methods 81, 3142, 1985; Cote et al., Proc. Natl. Acad. Sci. 80,2026-30, 1983; Cole et al., Mol. Cell. Biol. 62, 109-20, 1984).

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (Morrison et al., Proc. Natl. Acad.Sci. 81, 6851-55, 1984; Neuberger et al., Nature 312, 604-08, 1984;Takeda et al., Nature 314, 452-54, 1985). Monoclonal and otherantibodies also can be “humanized” to prevent a patient from mounting animmune response against the antibody when it is used therapeutically.Such antibodies may be sufficiently similar in sequence to humanantibodies to be used directly in therapy or may require alteration of afew key residues. Sequence differences between rodent antibodies andhuman sequences can be minimized by replacing residues which differ fromthose in the human sequences by site directed mutagenesis of individualresidues or by grating of entire complementarity determining regions.

Alternatively, humanized antibodies can be produced using recombinantmethods, as described below. Antibodies which specifically bind to aparticular antigen can contain antigen binding sites which are eitherpartially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.Human monoclonal antibodies can be prepared in vitro as described inSimmons et al., PLoS Medicine 4(5), 928-36, 2007.

Alternatively, techniques described for the production of single chainantibodies can be adapted using methods known in the art to producesingle chain antibodies which specifically bind to a particular antigen.Antibodies with related specificity, but of distinct idiotypiccomposition, can be generated by chain shuffling from randomcombinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88,11120-23, 1991).

Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template(Thirion et al., Eur. J. Cancer Prev. 5, 507-11, 1996). Single-chainantibodies can be mono- or bispecific, and can be bivalent ortetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught, for example, in Coloma & Morrison, Nat.Biotechnol. 15, 159-63, 1997. Construction of bivalent, bispecificsingle-chain antibodies is taught in Mallender & Voss, J. Biol. Chem.269, 199-206, 1994.

A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology (Verhaar et al., Int.J. Cancer 61, 497-501, 1995; Nicholls et al., J. Immunol. Meth. 165,81-91, 1993).

Antibodies which specifically bind to a GAS57 antigen also can beproduced by inducing in vivo production in the lymphocyte population orby screening immunoglobulin libraries or panels of highly specificbinding reagents as disclosed in the literature (Orlandi et al., Proc.Natl. Acad. Sci. 86, 3833 3837, 1989; Winter et al., Nature 349, 293299, 1991).

Chimeric antibodies can be constructed as disclosed in WO 93/03151.Binding proteins which are derived from immunoglobulins and which aremultivalent and multispecific, such as the “diabodies” described in WO94/13804, also can be prepared.

Antibodies can be purified by methods well known in the art. Forexample, antibodies can be affinity purified by passage over a column towhich the relevant antigen is bound. The bound antibodies can then beeluted from the column using a buffer with a high salt concentration.

Pharmaceutical Compositions

The invention also provides compositions for use as medicaments (e.g.,as immunogenic compositions or vaccines). Compositions of the inventionare useful for preventing and/or treating disease caused as a result ofS. pyogenes infection and comprise at least one active agent, which canbe a polypeptide, a nucleic acid molecule, or an antibody. Said diseasemay be, for example, bacteremia, meningitis, puerperal fever, scarletfever, erysipelas, pharyngitis, impetigo, necrotizing fasciitis,myositis or toxic shock syndrome.

Pharmaceutical compositions according to the invention may be usedeither prophylactically or therapeutically, but will typically beprophylactic. Accordingly, the invention includes a method for thetherapeutic or prophylactic treatment of a Streptococcus pyogenesinfection. The animal is preferably a mammal, most preferably a human.The methods involve administering to the animal a therapeutic orprophylactic amount of the immunogenic compositions of the invention.The invention also provides the immunogenic compositions of theinvention for the therapeutic or prophylactic treatment of aStreptococcus pyogenes infection in an animal.

Compositions containing mutant a GAS57 antigen or a nucleic acidmolecule encoding a mutant GAS57 antigen are preferably immunogeniccompositions, and are more preferably vaccine compositions. The pH ofsuch compositions preferably is between 6 and 8, preferably about 7. ThepH can be maintained by the use of a buffer. The composition can besterile and/or pyrogen free. The composition can be isotonic withrespect to human tissue (e.g., blood).

Some compositions of the invention comprise one or more mutant GAS57antigens as described herein. Other compositions of the inventioncomprise one or more nucleic acid molecules which encodes the mutantGAS57 antigen(s) and, optionally, other antigens which can be includedin the composition (see below). See, e.g., Robinson & Torres (1997)Seminars in Immunology 9:271-283; Donnelly et al. (1997) Ann. RevImmunol 15:617-648; Scott-Taylor & Dalgleish (2000) Expert Opin InvestigDrugs 9:471-480; Apostolopoulos & Plebanski (2000) Curr Opin Mol Ther2:441-447; Ilan (1999) Curr Opin Mol Ther 1:116-120; Dubensky et al.(2000) Mol Med 6:723-732; Robinson & Pertmer (2000) Adv Virus Res55:1-74; Donnelly et al. (2000) Am J Respir Crit. Care Med 162(4 Pt2):S190-193; Davis (1999) Mt. Sinai J. Med. 66:84-90. Typically thenucleic acid molecule is a DNA molecule, e.g., in the form of a plasmid.

Still other compositions of the invention comprise at least one antibodywhich specifically binds to a wild-type GAS57 antigen as described aboveor a nucleic acid molecule which encodes such an antibody.

In some embodiments, compositions of the invention can comprise morethan one type of active agent (e.g., a polypeptide antigen and a nucleicacid molecule; a polypeptide antigen and an antibody; a nucleic acidmolecule and an antibody; a polypeptide antigen, a nucleic acidmolecule, and an antibody).

In some embodiments, compositions of the invention can include one ormore additional active agents. Such agents include, but are not limitedto, (a) another mutant GAS57 antigen of the invention, (b) a polypeptideantigen which is useful in a pediatric vaccine, (c) a polypeptideantigen which is useful in a vaccine for elderly or immunocompromisedindividuals, (d) a nucleic acid molecule encoding (a)-(c), and anantibody which specifically binds to (a)-(c).

Additional Antigens

Compositions of the invention may be administered in conjunction withone or more antigens for use in therapeutic or prophylactic methods ofthe present invention. Preferred antigens include those listed below.Additionally, the compositions of the present invention may be used totreat or prevent infections caused by any of the below-listed pathogens.In addition to combination with the antigens described below, thecompositions of the invention may also be combined with an adjuvant asdescribed herein.

Antigens for use with the invention include, but are not limited to, oneor more of the following antigens set forth below, or antigens derivedfrom one or more of the pathogens set forth below:

A. Bacterial Antigens

Bacterial antigens suitable for use in the invention include proteins,polysaccharides, lipopolysaccharides, and outer membrane vesicles whichmay be isolated, purified or derived from a bacteria. In addition,bacterial antigens may include bacterial lysates and inactivatedbacterial formulations. Bacteria antigens may be produced by recombinantexpression. Bacterial antigens preferably include epitopes which areexposed on the surface of the bacteria during at least one stage of itslife cycle. Bacterial antigens are preferably conserved across multipleserotypes. Bacterial antigens include antigens derived from one or moreof the bacteria set forth below as well as the specific antigensexamples identified below.

Neisseria meningitidis: Meningitides antigens may include proteins (suchas those identified in References 1-7), saccharides (including apolysaccharide, oligosaccharide or lipopolysaccharide), orouter-membrane vesicles (References 8, 9, 10, 11) purified or derivedfrom N. meningitides serogroup such as A, C, W135, Y, and/or B.Meningitides protein antigens may be selected from adhesions,autotransporters, toxins, Fe acquisition proteins, and membraneassociated proteins (preferably integral outer membrane protein).

Streptococcus pneumoniae: Streptococcus pneumoniae antigens may includea saccharide (including a polysaccharide or an oligosaccharide) and/orprotein from Streptococcus pneumoniae. Saccharide antigens may beselected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Protein antigens maybe selected from a protein identified, in WO 98/18931, WO 98/18930, U.S.Pat. No. 6,699,703, U.S. Pat. No. 6,800,744, WO 97/43303, and WO97/37026. Streptococcus pneumoniae proteins may be selected from thePoly Histidine Triad family (PhtX), the Choline Binding Protein family(CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytXtruncate chimeric proteins, pneumolysin (Ply), PspA, PsaA, Sp128, Sp101,Sp130, Sp125 or Sp133.

Streptococcus pyogenes (Group A Streptococcus): Group A Streptococcusantigens may include a protein identified in WO 02/34771 or WO2005/032582 (including GAS 40), fusions of fragments of GAS M proteins(including those described in WO 02/094851, and Dale, Vaccine (1999)17:193-200, and Dale, Vaccine 14(10): 944-948), fibronectin bindingprotein (Sfb1), Streptococcal heme-associated protein (Shp), andStreptolysin S (SagA). Other Group A Streptococcus antigens include, butare not limited to, GAS25 (Spy0167; gi13621460) GAS39 (Spy0266;gi13621542), GAS40 (Spy0269; gi13621545), GAS42 (Spy0287; gi13621559),GAS45 (M5005_Spy0249; gi71910063), GAS58 (Spy0430; gi13621663), GAS84(SPy1274; 13622398), GAS95 (SPy1733; 13622787), GAS117 (Spy0448;gi13621679), GAS130 (Spy0591; gi13621794), GAS137 (Spy0652; gi13621842),GAS159 (Spy1105; gi13622244), GAS193 (Spy2025; gi3623029), GAS202(Spy1309; gi13622431), GAS217 (Spy0925, gi1362208), GAS236 (Spy1126;gi13622264), GAS253 (Spy1524; gi13622611), GAS277 (Spy1939; gi13622962),GAS294 (Spy1173; gi13622306), GAS309 (Spy0124; gi13621426), GAS366(Spy1525; gi13622612), GAS372 (Spy1625; gi13622698), GAS384 (Spy1874;gi13622908), GAS389 (Spy1981; gi13622996), GAS504 (Spy1751; gi13622806),GAS509 (Spy1618; gi13622692), GAS290 (SPy1959; gi13622978), GAS511(Spy1743; gi13622798), GAS527 (Spy1204; gi3622332), GAS529 (Spy1280;gi3622403), and GAS533 (Spy1877; gi13622912), GAS68 (Spy0163;gi13621456), GAS84 (Spy1274; gi13622398), GAS88 (Spy1361; gi13622470),GAS89 (Spy1390; gi13622493), GAS98 (Spy1882; gi13622916), GAS99(Spy1979; gi13622993), GAS102 (Spy2016, gi13623025), GAS146 (Spy0763;gi13621942), GAS195 (Spy2043; gi13623043), GAS561 (Spy1134; gi13622269),GAS179 (Spy1718, gi13622773) and GAS681 (spy1152; gi1362228).

Moraxella catarrhalis: Moraxella antigens include antigens identified inWO 02/18595 and WO 99/58562, outer membrane protein antigens (HMW-OMP),C-antigen, and/or LPS.

Bordetella pertussis: Pertussis antigens include petussis holotoxin (PT)and filamentous haemagglutinin (FHA) from B. pertussis, optionally alsocombination with pertactin and/or agglutinogens 2 and 3 antigen.

Staphylococcus aureus: Staphylococcus aureus antigens include S. aureustype 5 and 8 capsular polysaccharides optionally conjugated to nontoxicrecombinant Pseudomonas aeruginosa exotoxin A, such as StaphVAX™, orantigens derived from surface proteins, invasins (leukocidin, kinases,hyaluronidase), surface factors that inhibit phagocytic engulfment(capsule, Protein A), carotenoids, catalase production, Protein A,coagulase, clotting factor, and/or membrane-damaging toxins (optionallydetoxified) that lyse eukaryotic cell membranes (hemolysins, leukotoxin,leukocidin).

Staphylococcus epidermis: S. epidermidis antigens includeslime-associated antigen (SAA).

Clostridium tetani (Tetanus): Tetanus antigens include tetanus toxoid(TT), preferably used as a carrier protein in conjunction/conjugatedwith the compositions of the present invention.

Cornynebacterium diphtheriae (Diphtheria): Diphtheria antigens includediphtheria toxin, preferably detoxified, such as CRM197. Additionallyantigens capable of modulating, inhibiting or associated with ADPribosylation are contemplated forcombination/co-administration/conjugation with the compositions of thepresent invention. The diphtheria toxoids may be used as carrierproteins.

Haemophilus influenzae B (Hib): Hib antigens include a Hib saccharideantigen.

Pseudomonas aeruginosa: Pseudomonas antigens include endotoxin A, Wzzprotein, P. aeruginosa LPS, more particularly LPS isolated from PAO1 (O5serotype), and/or Outer Membrane Proteins, including Outer MembraneProteins F (OprF) (Infect Immun. 2001 May; 69(5): 3510-3515).

Legionella pneumophila. Bacterial antigens may be derived fromLegionella pneumophila.

Streptococcus agalactiae (Group B Streptococcus): Group B Streptococcusantigens include a protein or saccharide antigen identified in WO02/34771, WO 03/093306, WO 04/041157, or WO 2005/002619 (includingproteins GBS 80, GBS 104, GBS 276 and GBS 322, and including saccharideantigens derived from serotypes Ia, Ib, Ia/c, II, III, IV, V, VI, VIIand VIII).

Neiserria gonorrhoeae: Gonorrhoeae antigens include Por (or porin)protein, such as PorB (see Zhu et al., Vaccine (2004) 22:660-669), atransferring binding protein, such as TbpA and TbpB (See Price et al.,Infection and Immunity (2004) 71(1):277-283), a opacity protein (such asOpa), a reduction-modifiable protein (Rmp), and outer membrane vesicle(OMV) preparations (see Plante et al., J Infectious Disease (2000)182:848-855), also see e.g. WO99/24578, WO99/36544, WO99/57280,WO02/079243).

Chlamydia trachomatis: Chlamydia trachomatis antigens include antigensderived from serotypes A, B, Ba and C (agents of trachoma, a cause ofblindness), serotypes L1, L2 & L3 (associated with Lymphogranulomavenereum), and serotypes, D-K. Chlamydia trachomas antigens may alsoinclude an antigen identified in WO 00/37494, WO 03/049762, WO03/068811, or WO 05/002619, including PepA (CT045), LcrE (CT089), ArtJ(CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA(CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), and MurG(CT761).

Treponema pallidum (Syphilis): Syphilis antigens include TmpA antigen.

Haemophilus ducreyi (causing chancroid): Ducreyi antigens include outermembrane protein (DsrA).

Enterococcus faecalis or Enterococcus faecium: Antigens include atrisaccharide repeat or other Enterococcus derived antigens provided inU.S. Pat. No. 6,756,361.

Helicobacter pylori: H. pylori antigens include Cag, Vac, Nap, HopX,HopY and/or urease antigen.

Staphylococcus saprophyticus: Antigens include the 160 kDa hemagglutininof S. saprophyticus antigen.

Yersinia enterocolitica antigens include LPS (Infect Immun. 2002 August;70(8): 4414).

E. coli: E. coli antigens may be derived from enterotoxigenic E. coli(ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli(DAEC), enteropathogenic E. coli (EPEC), and/or enterohemorrhagic E.coli (EHEC).

Bacillus anthracis (anthrax): B. anthracis antigens are optionallydetoxified and may be selected from A-components (lethal factor (LF) andedema factor (EF)), both of which can share a common B-component knownas protective antigen (PA).

Yersinia pestis (plague): Plague antigens include F1 capsular antigen(Infect Immun. 2003 January; 71(1)): 374-383, LPS (Infect Immun. 1999October; 67(10): 5395), Yersinia pestis V antigen (Infect Immun. 1997November; 65(11): 4476-4482).

Mycobacterium tuberculosis: Tuberculosis antigens include lipoproteins,LPS, BCG antigens, a fusion protein of antigen 85B (Ag85B) and/or ESAT-6optionally formulated in cationic lipid vesicles (Infect Immun. 2004October; 72(10): 6148), Mycobacterium tuberculosis (Mtb) isocitratedehydrogenase associated antigens (Proc Natl Acad Sci USA. 2004 Aug. 24;101(34): 12652), and/or MPT51 antigens (Infect Immun. 2004 July; 72(7):3829).

Rickettsia: Antigens include outer membrane proteins, including theouter membrane protein A and/or B (OmpB) (Biochim Biophys Acta. 2004Nov. 1; 1702(2):145), LPS, and surface protein antigen (SPA) (J.Autoimmun. 1989 June; 2 Suppl:81).

Listeria monocytogenes. Bacterial antigens may be derived from Listeriamonocytogenes.

Chlamydia pneumoniae: Antigens include those identified in WO 02/02606.

Vibrio cholerae: Antigens include proteinase antigens, LPS, particularlylipopolysaccharides of Vibrio cholerae II, O1 Inaba O-specificpolysaccharides, V. cholera O139, antigens of IEM108 vaccine (InfectImmun. 2003 October; 71(10):5498-504), and/or Zonula occludens toxin(Zot).

Salmonella typhi (typhoid fever): Antigens include capsularpolysaccharides preferably conjugates (Vi, i.e. vax-TyVi).

Borrelia burgdorferi (Lyme disease): Antigens include lipoproteins (suchas OspA, OspB, OspC and OspD), other surface proteins such asOspE-related proteins (Erps), decorin-binding proteins (such as DbpA),and antigenically variable VI proteins, such as antigens associated withP39 and P13 (an integral membrane protein, Infect Immun. 2001 May;69(5): 3323-3334), VlsE Antigenic Variation Protein (J Clin Microbiol.1999 December; 37(12): 3997).

Porphyromonas gingivalis: Antigens include P. gingivalis outer membraneprotein (OMP).

Klebsiella: Antigens include an OMP, including OMP A, or apolysaccharide optionally conjugated to tetanus toxoid.

Further bacterial antigens of the invention may be capsular antigens,polysaccharide antigens or protein antigens of any of the above. Furtherbacterial antigens may also include an outer membrane vesicle (OMV)preparation. Additionally, antigens include live, attenuated, and/orpurified versions of any of the aforementioned bacteria. The antigens ofthe present invention may be derived from gram-negative or gram-positivebacteria. The antigens of the present invention may be derived fromaerobic or anaerobic bacteria.

Additionally, any of the above bacterial-derived saccharides(polysaccharides, LPS, LOS or oligosaccharides) can be conjugated toanother agent or antigen, such as a carrier protein (for exampleCRM197). Such conjugation may be direct conjugation effected byreductive amination of carbonyl moieties on the saccharide to aminogroups on the protein, as provided in U.S. Pat. No. 5,360,897 and Can JBiochem Cell Biol. 1984 May; 62(5):270-5. Alternatively, the saccharidescan be conjugated through a linker, such as, with succinamide or otherlinkages provided in Bioconjugate Techniques, 1996 and CRC, Chemistry ofProtein Conjugation and Cross-Linking, 1993.

B. Viral Antigens

Viral antigens suitable for use in the invention include inactivated (orkilled) virus, attenuated virus, split virus formulations, purifiedsubunit formulations, viral proteins which may be isolated, purified orderived from a virus, and Virus Like Particles (VLPs). Viral antigensmay be derived from viruses propagated on cell culture or othersubstrate. Alternatively, viral antigens may be expressed recombinantly.Viral antigens preferably include epitopes which are exposed on thesurface of the virus during at least one stage of its life cycle. Viralantigens are preferably conserved across multiple serotypes or isolates.Viral antigens include antigens derived from one or more of the virusesset forth below as well as the specific antigens examples identifiedbelow.

Orthomyxovirus: Viral antigens may be derived from an Orthomyxovirus,such as Influenza A, B and C. Orthomyxovirus antigens may be selectedfrom one or more of the viral proteins, including hemagglutinin (HA),neuraminidase (NA), nucleoprotein (NP), matrix protein (M1), membraneprotein (M2), one or more of the transcriptase components (PB1, PB2 andPA). Preferred antigens include HA and NA.

Influenza antigens may be derived from interpandemic (annual) flustrains. Alternatively influenza antigens may be derived from strainswith the potential to cause pandemic a pandemic outbreak (i.e.,influenza strains with new haemagglutinin compared to the haemagglutininin currently circulating strains, or influenza strains which arepathogenic in avian subjects and have the potential to be transmittedhorizontally in the human population, or influenza strains which arepathogenic to humans).

Paramyxoviridae viruses: Viral antigens may be derived fromParamyxoviridae viruses, such as Pneumoviruses (RSV), Paramyxoviruses(PIV) and Morbilliviruses (Measles).

Pneumovirus: Viral antigens may be derived from a Pneumovirus, such asRespiratory syncytial virus (RSV), Bovine respiratory syncytial virus,Pneumonia virus of mice, and Turkey rhinotracheitis virus. Preferably,the Pneumovirus is RSV. Pneumovirus antigens may be selected from one ormore of the following proteins, including surface proteins Fusion (F),Glycoprotein (G) and Small Hydrophobic protein (SH), matrix proteins Mand M2, nucleocapsid proteins N, P and L and nonstructural proteins NS 1and NS2. Preferred Pneumovirus antigens include F, G and M. See e.g., JGen Virol. 2004 November; 85(Pt 11):3229). Pneumovirus antigens may alsobe formulated in or derived from chimeric viruses. For example, chimericRSV/PIV viruses may comprise components of both RSV and PIV.

Paramyxovirus: Viral antigens may be derived from a Paramyxovirus, suchas Parainfluenza virus types 1-4 (PIV), Mumps, Sendai viruses, Simianvirus 5, Bovine parainfluenza virus and Newcastle disease virus.Preferably, the Paramyxovirus is PIV or Mumps. Paramyxovirus antigensmay be selected from one or more of the following proteins:Hemagglutinin-Neuraminidase (HN), Fusion proteins F 1 and F2,Nucleoprotein (NP), Phosphoprotein (P), Large protein (L), and Matrixprotein (M). Preferred Paramyxovirus proteins include HN, F1 and F2.Paramyxovirus antigens may also be formulated in or derived fromchimeric viruses. For example, chimeric RSV/PIV viruses may comprisecomponents of both RSV and Ply. Commercially available mumps vaccinesinclude live attenuated mumps virus, in either a monovalent form or incombination with measles and rubella vaccines (MMR).

Morbillivirus: Viral antigens may be derived from a Morbillivirus, suchas Measles. Morbillivirus antigens may be selected from one or more ofthe following proteins: hemagglutinin (H), Glycoprotein (G), Fusionfactor (F), Large protein (L), Nucleoprotein (NP), Polymerasephosphoprotein (P), and Matrix (M). Commercially available measlesvaccines include live attenuated measles virus, typically in combinationwith mumps and rubella (MMR).

Picornavirus: Viral antigens may be derived from Picornaviruses, such asEnteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses andAphthoviruses. Antigens derived from Enteroviruses, such as Poliovirusare preferred.

Enterovirus: Viral antigens may be derived from an Enterovirus, such asPoliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24,Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus) types 1 to 9, 11to 27 and 29 to 34 and Enterovirus 68 to 71. Preferably, the Enterovirusis poliovirus. Enterovirus antigens are preferably selected from one ormore of the following Capsid proteins VP1, VP2, VP3 and VP4.Commercially available polio vaccines include Inactivated Polio Vaccine(IPV) and Oral poliovirus vaccine (OPV).

Heparnavirus: Viral antigens may be derived from an Heparnavirus, suchas Hepatitis A virus (HAV). Commercially available HAV vaccines includeinactivated HAV vaccine.

Togavirus: Viral antigens may be derived from a Togavirus, such as aRubivirus, an Alphavirus, or an Arterivirus. Antigens derived fromRubivirus, such as Rubella virus, are preferred. Togavirus antigens maybe selected from E1, E2, E3, C, NSP-1, NSPO-2, NSP-3 or NSP-4. Togavirusantigens are preferably selected from E1, E2 or E3. Commerciallyavailable Rubella vaccines include a live cold-adapted virus, typicallyin combination with mumps and measles vaccines (MMR).

Flavivirus: Viral antigens may be derived from a Flavivirus, such asTick-borne encephalitis (TBE), Dengue (types 1, 2, 3 or 4), YellowFever, Japanese encephalitis, West Nile encephalitis, St. Louisencephalitis, Russian spring-summer encephalitis, Powassan encephalitis.Flavivirus antigens may be selected from PrM, M, C, E, NS-1, NS-2a,NS2b, NS3, NS4a, NS4b, and NS5. Flavivirus antigens are preferablyselected from PrM, M and E. Commercially available TBE vaccine includeinactivated virus vaccines.

Pestivirus: Viral antigens may be derived from a Pestivirus, such asBovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Borderdisease (BDV).

Hepadnavirus: Viral antigens may be derived from a Hepadnavirus, such asHepatitis B virus. Hepadnavirus antigens may be selected from surfaceantigens (L, M and S), core antigens (HBc, HBe). Commercially availableHBV vaccines include subunit vaccines comprising the surface antigen Sprotein.

Hepatitis C virus: Viral antigens may be derived from a Hepatitis Cvirus (HCV). HCV antigens may be selected from one or more of E1, E2,E1/E2, NS345 polyprotein, NS 345-core polyprotein, core, and/or peptidesfrom the nonstructural regions (Houghton et al., Hepatology (1991)14:381).

Rhabdovirus: Viral antigens may be derived from a Rhabdovirus, such as aLyssavirus (Rabies virus) and Vesiculovirus (VSV). Rhabdovirus antigensmay be selected from glycoprotein (G), nucleoprotein (N), large protein(L), nonstructural proteins (NS). Commercially available Rabies virusvaccine comprise killed virus grown on human diploid cells or fetalrhesus lung cells.

Caliciviridae; Viral antigens may be derived from Calciviridae, such asNorwalk virus, and Norwalk-like Viruses, such as Hawaii Virus and SnowMountain Virus.

Coronavirus: Viral antigens may be derived from a Coronavirus, SARS,Human respiratory coronavirus, Avian infectious bronchitis (IBV), Mousehepatitis virus (MHV), and Porcine transmissible gastroenteritis virus(TGEV). Coronavirus antigens may be selected from spike (S), envelope(E), matrix (M), nucleocapsid (N), and Hemagglutinin-esteraseglycoprotein (HE). Preferably, the Coronavirus antigen is derived from aSARS virus. SARS viral antigens are described in WO 04/92360;

Retrovirus: Viral antigens may be derived from a Retrovirus, such as anOncovirus, a Lentivirus or a Spumavirus. Oncovirus antigens may bederived from HTLV-1, HTLV-2 or HTLV-5. Lentivirus antigens may bederived from HIV-1 or HIV-2. Retrovirus antigens may be selected fromgag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. HIV antigensmay be selected from gag (p24gag and p55gag), env (gp160 and gp41), pol,tat, nef, rev vpu, miniproteins, (preferably p55 gag and gp140v delete).HIV antigens may be derived from one or more of the following strains:HIVIIIb, HIVSF2, HIVLAV, HILAI, HIVMN, HIV-1CM235, HIV-1US4.

Reovirus: Viral antigens may be derived from a Reovirus, such as anOrthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus. Reovirusantigens may be selected from structural proteins λ1, λ2, 80 3, μ1, μ2,σ1, σ2, or σ3, or nonstructural proteins σNS, μNS, or a σ1s. PreferredReovirus antigens may be derived from a Rotavirus. Rotavirus antigensmay be selected from VP1, VP2, VP3, VP4 (or the cleaved product VP5 andVP8), NSP 1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5. Preferred Rotavirusantigens include VP4 (or the cleaved product VP5 and VP8), and VP7.

Parvovirus: Viral antigens may be derived from a Parvovirus, such asParvovirus B 19. Parvovirus antigens may be selected from VP-1, VP-2,VP-3, NS-1 and NS-2. Preferably, the Parvovirus antigen is capsidprotein VP-2.

Delta hepatitis virus (HDV): Viral antigens may be derived HDV,particularly δ-antigen from HDV (see, e.g., U.S. Pat. No. 5,378,814).

Hepatitis E virus (HEV): Viral antigens may be derived from HEV.

Hepatitis G virus (HGV): Viral antigens may be derived from HGV.

Human Herpesvirus: Viral antigens may be derived from a HumanHerpesvirus, such as

Herpes Simplex Viruses (RSV), Varicella-zoster virus (VZV), Epstein-Barrvirus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), HumanHerpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8). Human Herpesvirusantigens may be selected from immediate early proteins (α), earlyproteins (β), and late proteins (γ). HSV antigens may be derived fromHSV-1 or HSV-2 strains. HSV antigens may be selected from glycoproteinsgB, gC, gD and gH, fusion protein (gB), or immune escape proteins (gC,gE, or gI). VZV antigens may be selected from core, nucleocapsid,tegument, or envelope proteins. A live attenuated VZV vaccine iscommercially available. EBV antigens may be selected from early antigen(EA) proteins, viral capsid antigen (VCA), and glycoproteins of themembrane antigen (MA). CMV antigens may be selected from capsidproteins, envelope glycoproteins (such as gB and gH), and tegumentproteins

Papovaviruses: Antigens may be derived from Papovaviruses, such asPapillomaviruses and Polyomaviruses. Papillomaviruses include HPVserotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47,51, 57, 58, 63 and 65. Preferably, HPV antigens are derived fromserotypes 6, 11, 16 or 18. HPV antigens may be selected from capsidproteins (L1) and (L2), or E1-E7, or fusions thereof. HPV antigens arepreferably formulated into virus-like particles (VLPs). Polyomyavirusviruses include BK virus and JK virus. Polyomavirus antigens may beselected from VP1, VP2 or VP3.

Further provided are antigens, compositions, methods, and microbesincluded in Vaccines, 4th Edition (Plotkin and Orenstein ed. 2004);Medical Microbiology 4th Edition (Murray et al. ed. 2002); Virology, 3rdEdition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B.N. Fields and D. M. Knipe, eds. 1991), which are contemplated inconjunction with the compositions of the present invention.

C. Fungal Antigens

Fungal antigens for use in the invention may be derived from one or moreof the fungi set forth below.

Fungal antigens may be derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis,Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T. verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme.

Fungal pathogens may be derived from Aspergillus fumigatus, Aspergillusflavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus,Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus,Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candidatropicalis, Candida glabrata, Candida krusei, Candida parapsilosis,Candida stellatoidea, Candida kusei, Candida parakwsei, Candidalusitaniae, Candida pseudotropicalis, Candida guilliermondi,Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis,Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum,Klebsiella pneumoniae, Paracoccidioides brasiliensis, Pneumocystiscarinii, Pythiumn insidiosum, Pityrosporum ovale, Saccharomycescerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporiumapiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasmagondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp.,Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp.,Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamellaspp, Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporiumspp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp,Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp.

Processes for producing a fungal antigens are well known in the art (seeU.S. Pat. No. 6,333,164). In a preferred method a solubilized fractionextracted and separated from an insoluble fraction obtainable fromfungal cells of which cell wall has been substantially removed or atleast partially removed, characterized in that the process comprises thesteps of: obtaining living fungal cells; obtaining fungal cells of whichcell wall has been substantially removed or at least partially removed;bursting the fungal cells of which cell wall has been substantiallyremoved or at least partially removed; obtaining an insoluble fraction;and extracting and separating a solubilized fraction from the insolublefraction.

D. STD Antigens

The compositions of the invention may include one or more antigensderived from a sexually transmitted disease (STD). Such antigens mayprovide for prophylactic or therapy for STDs such as chlamydia, genitalherpes, hepatits (such as HCV), genital warts, gonorrhoea, syphilisand/or chancroid (See, WO00/15255). Antigens may be derived from one ormore viral or bacterial STDs. Viral STD antigens for use in theinvention may be derived from, for example, HIV, herpes simplex virus(HSV-1 and HSV-2), human papillomavirus (HPV), and hepatitis (HCV).Bacterial STD antigens for use in the invention may be derived from, forexample, Neiserria gonorrhoeae, Chlamydia trachomatis, Treponemapallidum, Haemophilus ducreyi, E. coli, and Streptococcus agalactiae.Examples of specific antigens derived from these pathogens are describedabove.

E. Respiratory Antigens

The compositions of the invention may include one or more antigensderived from a pathogen which causes respiratory disease. For example,respiratory antigens may be derived from a respiratory virus such asOrthomyxoviruses (influenza), Pneumovirus (RSV), Paramyxovirus (PIV),Morbillivirus (measles), Togavirus (Rubella), VZV, and Coronavirus(SARS). Respiratory antigens may be derived from a bacteria which causesrespiratory disease, such as Streptococcus pneumoniae, Pseudomonasaeruginosa, Bordetella pertussis, Mycobacterium tuberculosis, Mycoplasmapneumoniae, Chlamydia pneumoniae, Bacillus anthracia, and Moraxellacatarrhalis. Examples of specific antigens derived from these pathogensare described above.

F. Pediatric Vaccine Antigens

The compositions of the invention may include one or more antigenssuitable for use in pediatric subjects. Pediatric subjects are typicallyless than about 3 years old, or less than about 2 years old, or lessthan about 1 years old. Pediatric antigens may be administered multipletimes over the course of 6 months, 1, 2 or 3 years. Pediatric antigensmay be derived from a virus which may target pediatric populationsand/or a virus from which pediatric populations are susceptible toinfection. Pediatric viral antigens include antigens derived from one ormore of Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus(PIV and Mumps), Morbillivirus (measles), Togavirus (Rubella),Enterovirus (polio), HBV, Coronavirus (SARS), and Varicella-zoster virus(VZV), Epstein Barr virus (EBV). Pediatric bacterial antigens includeantigens derived from one or more of Streptococcus pneumoniae, Neisseriameningitides, Streptococcus pyogenes (Group A Streptococcus), Moraxellacatarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridiumtetani (Tetanus), Corynebacterium diphtheriae (Diphtheria), Haemophilusinfluenzae B (Hib), Pseudomonas aeruginosa, Streptococcus agalactiae(Group B Streptococcus), and E. coli. Examples of specific antigensderived from these pathogens are described above.

G. Antigens Suitable for Use in Elderly or Immunocompromised Individuals

The compositions of the invention may include one or more antigenssuitable for use in elderly or immunocompromised individuals. Suchindividuals may need to be vaccinated more frequently, with higher dosesor with adjuvanted formulations to improve their immune response to thetargeted antigens. Antigens which may be targeted for use in Elderly orImmunocompromised individuals include antigens derived from one or moreof the following pathogens: Neisseria meningitides, Streptococcuspneumoniae, Streptococcus pyogenes (Group A Streptococcus), Moraxellacatarrhalis, Bordetella pertussis, Staphylococcus aureus, Staphylococcusepidermis, Clostridium tetani (Tetanus), Cornynebacterium diphtheriae(Diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa,Legionella pneumophila, Streptococcus agalactiae (Group BStreptococcus), Enterococcus faecalis, Helicobacter pylori, Clamydiapneumoniae, Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus(PIV and Mumps), Morbillivirus (measles), Togavirus (Rubella),Enterovirus (polio), HBV, Coronavirus (SARS), Varicella-zoster virus(VZV), Epstein Barr virus (EBV), Cytomegalovirus (CMV). Examples ofspecific antigens derived from these pathogens are described above.

H. Antigens Suitable for Use in Adolescent Vaccines

The compositions of the invention may include one or more antigenssuitable for use in adolescent subjects. Adolescents may be in need of aboost of a previously administered pediatric antigen. Pediatric antigenswhich may be suitable for use in adolescents are described above. Inaddition, adolescents may be targeted to receive antigens derived froman STD pathogen in order to ensure protective or therapeutic immunitybefore the beginning of sexual activity. STD antigens which may besuitable for use in adolescents are described above.

I. Antigen Formulations

In other aspects of the invention, methods of producing microparticleshaving adsorbed antigens are provided. The methods comprise: (a)providing an emulsion by dispersing a mixture comprising (i) water, (ii)a detergent, (iii) an organic solvent, and (iv) a biodegradable polymerselected from the group consisting of a poly(α-hydroxy acid), apolyhydroxy butyric acid, a polycaprolactone, a polyorthoester, apolyanhydride, and a polycyanoacrylate. The polymer is typically presentin the mixture at a concentration of about 1% to about 30% relative tothe organic solvent, while the detergent is typically present in themixture at a weight-to-weight detergent-to-polymer ratio of from about0.00001:1 to about 0.1:1 (more typically about 0.0001:1 to about 0.1:1,about 0.001:1 to about 0.1:1, or about 0.005:1 to about 0.1:1); (b)removing the organic solvent from the emulsion; and (c) adsorbing anantigen on the surface of the microparticles. In certain embodiments,the biodegradable polymer is present at a concentration of about 3% toabout 10% relative to the organic solvent.

Microparticles for use herein will be formed from materials that aresterilizable, non-toxic and biodegradable. Such materials include,without limitation, poly(α-hydroxy acid), polyhydroxybutyric acid,polycaprolactone, polyorthoester, polyanhydride, PACA, andpolycyanoacrylate. Preferably, microparticles for use with the presentinvention are derived from a poly(α-hydroxy acid), in particular, from apoly(lactide) (“PLA”) or a copolymer of D,L-lactide and glycolide orglycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or“PLGA”), or a copolymer of D,L-lactide and caprolactone. Themicroparticles may be derived from any of various polymeric startingmaterials which have a variety of molecular weights and, in the case ofthe copolymers such as PLG, a variety of lactide:glycolide ratios, theselection of which will be largely a matter of choice, depending in parton the coadministered macromolecule. These parameters are discussed morefully below.

Further antigens may also include an outer membrane vesicle (OMV)preparation.

Additional formulation methods and antigens (especially tumor antigens)are provided in U.S. patent Ser. No. 09/581,772.

J. Antigen References

The following references include antigens useful in conjunction with thecompositions of the present invention:

-   1 International patent application WO99/24578-   2 International patent application WO99/36544.-   3 International patent application WO99/57280.-   4 International patent application WO00/22430.-   5 Tettelin et al. (2000) Science 287:1809-1815.-   6 International patent application WO96/29412.-   7 Pizza et al. (2000) Science 287:1816-1820.-   8 PCT WO 01/52885.-   9 Bjune et al. (1991) Lancet 338(8775).-   10 Fuskasawa et al. (1999) Vaccine 17:2951-2958.-   11 Rosenqist et al. (1998) Dev. Biol. Strand 92:323-333.-   12 Constantino et al. (1992) Vaccine 10:691-698.-   13 Constantino et al. (1999) Vaccine 17:1251-1263.-   14 Watson (2000) Pediatr Infect Dis J 19:331-332.-   15 Rubin (20000) Pediatr Clin North Am 47:269-285,v.-   16 Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207.-   17 International patent application filed on 3 Jul. 2001 claiming    priority from GB-0016363.4; WO 02/02606; PCT IB/01/00166.-   18 Kalman et al. (1999) Nature Genetics 21:385-389.-   19 Read et al. (2000) Nucleic Acids Res 28:1397-406.-   20 Shirai et al. (2000) J. Infect. Dis 181(Suppl 3):S524-S527.-   21 International patent application WO99/27105.-   22 International patent application WO00/27994.-   23 International patent application WO00/37494.-   24 International patent application WO99/28475.-   25 Bell (2000) Pediatr Infect Dis J 19:1187-1188.-   26 Iwarson (1995) APMIS 103:321-326.-   27 Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.-   28 Hsu et al. (1999) Clin Liver Dis 3:901-915.-   29 Gastofsson et al. (1996) N. Engl. J. Med. 334-349-355.-   30 Rappuoli et al. (1991) TIBTECH 9:232-238.-   31 Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0.-   32 Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70.-   33 International patent application WO93/018150.-   34 International patent application WO99/53310.-   35 International patent application WO98/04702.-   36 Ross et al. (2001) Vaccine 19:135-142.-   37 Sutter et al. (2000) Pediatr Clin North Am 47:287-308.-   38 Zimmerman & Spann (1999) Am Fan Physician 59:113-118, 125-126.-   39 Dreensen (1997) Vaccine 15 Suppl” S2-6.-   40 MMWR Morb Mortal Wkly rep 1998 January 16:47(1):12, 9.-   41 McMichael (2000) Vaccine 19 Suppl 1:S101-107.-   42 Schuchat (1999) Lancer 353(9146):51-6.-   43 GB patent applications 0026333.5, 0028727.6 & 0105640.7.-   44 Dale (1999) Infect Disclin North Am 13:227-43, viii.-   45 Ferretti et al. (2001) PNAS USA 98: 4658-4663.-   46 Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages    1218-1219.-   47 Ramsay et al. (2001) Lancet 357(9251):195-196.-   48 Lindberg (1999) Vaccine 17 Suppl 2:S28-36.-   49 Buttery & Moxon (2000) J R Coil Physicians Long 34:163-168.-   50 Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii.-   51 Goldblatt (1998) J. Med. Microbiol. 47:663-567.-   52 European patent 0 477 508.-   53 U.S. Pat. No. 5,306,492.-   54 International patent application WO98/42721.-   55 Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326,    particularly vol. 10:48-114.-   56 Hermanson (1996) Bioconjugate Techniques ISBN: 012323368 &    012342335X.-   57 European patent application 0372501.-   58 European patent application 0378881.-   59 European patent application 0427347.-   60 International patent application WO93/17712.-   61 International patent application WO98/58668.-   62 European patent application 0471177.-   63 International patent application WO00/56360.-   64 International patent application WO00/67161.

The contents of all of the above cited patents, patent applications andjournal articles are incorporated by reference as if set forth fullyherein.

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier protein in order to enhance immunogenicity. SeeRamsay et al. (2001) Lancet 357(9251):195-196; Lindberg (1999) Vaccine17 Suppl 2:S28-36; Buttery & Moxon (2000) J R Coll Physicians Lond34:163-168; Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133,vii; Goldblatt (1998) J. Med. Microbiol. 47:563-567; European patent 0477 508; U.S. Pat. No. 5,306,492; WO98/42721; Conjugate Vaccines (eds.Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114; Hermanson(1996) Bioconjugate Techniques ISBN: 0123423368 or 012342335X. Preferredcarrier proteins are bacterial toxins or toxoids, such as diphtheria ortetanus toxoids. The CRM197 diphtheria toxoid is particularly preferred.

Other carrier polypeptides include the N. meningitidis outer membraneprotein (EP-A-0372501), synthetic peptides (EP-A-0378881 and EP-A0427347), heat shock proteins (WO 93/17712 and WO 94/03208), pertussisproteins (WO 98/58668 and EP A 0471177), protein D from H. influenzae(WO 00/56360), cytokines (WO 91/01146), lymphokines, hormones, growthfactors, toxin A or B from C. difficile (WO 00/61761), iron-uptakeproteins (WO 01/72337), etc. Where a mixture comprises capsularsaccharide from both serigraphs A and C, it may be preferred that theratio (w/w) of MenA saccharide:MenC saccharide is greater than 1 (e.g.,2:1, 3:1, 4:1, 5:1, 10:1 or higher). Different saccharides can beconjugated to the same or different type of carrier protein. Anysuitable conjugation reaction can be used, with any suitable linkerwhere necessary.

Toxic protein antigens may be detoxified where necessary e.g.,detoxification of pertussis toxin by chemical and/or genetic means.

Pharmaceutically Acceptable Carriers

Compositions of the invention will typically, in addition to thecomponents mentioned above, comprise one or more “pharmaceuticallyacceptable carriers.” These include any carrier which does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers typically are large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and lipid aggregates (such as oil droplets or liposomes). Such carriersare well known to those of ordinary skill in the art. A composition mayalso contain a diluent, such as water, saline, glycerol, etc.Additionally, an auxiliary substance, such as a wetting or emulsifyingagent, pH buffering substance, and the like, may be present. A thoroughdiscussion of pharmaceutically acceptable components is available inGennaro (2000) Remington: The Science and Practice of Pharmacy. 20thed., ISBN: 0683306472.

Immunoregulatory Agents

Adjuvants

Vaccines of the invention may be administered in conjunction with otherimmunoregulatory agents. In particular, compositions will usuallyinclude an adjuvant. Adjuvants for use with the invention include, butare not limited to, one or more of the following set forth below:

A. Mineral Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminum salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulfates, etc. (e.g. see chapters 8 & 9 of Vaccine Design . . . (1995)eds. Powell & Newman. ISBN: 030644867X. Plenum.), or mixtures ofdifferent mineral compounds (e.g. a mixture of a phosphate and ahydroxide adjuvant, optionally with an excess of the phosphate), withthe compounds taking any suitable form (e.g. gel, crystalline,amorphous, etc.), and with adsorption to the salt(s) being preferred.The mineral containing compositions may also be formulated as a particleof metal salt (WO00/23105).

Aluminum salts may be included in vaccines of the invention such thatthe dose of Al³⁺ is between 0.2 and 1.0 mg per dose.

In one embodiment the aluminum based adjuvant for use in the presentinvention is alum (aluminum potassium sulfate (AlK(SO₄)₂)), or an alumderivative, such as that formed in-situ by mixing an antigen inphosphate buffer with alum, followed by titration and precipitation witha base such as ammonium hydroxide or sodium hydroxide.

Another aluminum-based adjuvant for use in vaccine formulations of thepresent invention is aluminum hydroxide adjuvant (Al(OH)₃) orcrystalline aluminum oxyhydroxide (AlOOH), which is an excellentadsorbant, having a surface area of approximately 500 m²/g.Alternatively, aluminum phosphate adjuvant (AlPO₄) or aluminumhydroxyphosphate, which contains phosphate groups in place of some orall of the hydroxyl groups of aluminum hydroxide adjuvant is provided.Preferred aluminum phosphate adjuvants provided herein are amorphous andsoluble in acidic, basic and neutral media.

In another embodiment the adjuvant of the invention comprises bothaluminum phosphate and aluminum hydroxide. In a more particularembodiment thereof, the adjuvant has a greater amount of aluminumphosphate than aluminum hydroxide, such as a ratio of 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1 or greater than 9:1, by weight aluminumphosphate to aluminum hydroxide. More particular still, aluminum saltsin the vaccine are present at 0.4 to 1.0 mg per vaccine dose, or 0.4 to0.8 mg per vaccine dose, or 0.5 to 0.7 mg per vaccine dose, or about 0.6mg per vaccine dose.

Generally, the preferred aluminum-based adjuvant(s), or ratio ofmultiple aluminum-based adjuvants, such as aluminum phosphate toaluminum hydroxide is selected by optimization of electrostaticattraction between molecules such that the antigen carries an oppositecharge as the adjuvant at the desired pH. For example, aluminumphosphate adjuvant (isoelectric point=4) adsorbs lysozyme, but notalbumin at pH 7.4. Should albumin be the target, aluminum hydroxideadjuvant would be selected (iep 11.4). Alternatively, pretreatment ofaluminum hydroxide with phosphate lowers its isoelectric point, makingit a preferred adjuvant for more basic antigens.

B. Oil-Emulsions

Oil-emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 (5% squalene, 0.5%TWEEN®80 (polyoxyethylene sorbitan monooleate), and 0.5% SPAN®85(sorbitan trioleate)), formulated into submicron particles using amicrofluidizer). See W090/14837. See also, Podda, Vaccine (2001)I9:2673-2680; Frey et al., Vaccine (2003) 21:4234-4237. MF59 is used as theadjuvant in the FLUAD™ influenza virus trivalent subunit vaccine.

Particularly preferred adjuvants for use in the compositions aresubmicron oil-in-water emulsions. Preferred submicron oil-in-wateremulsions for use herein are squalene/water emulsions optionallycontaining varying amounts of MTP-PE, such as a submicron oil-in-wateremulsion containing 4-5% w/v squalene, 0.25-1.0% w/v TWEEN®80(polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% SPAN®85(sorbitan trioleate), and, optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-s-n-glycero-3-huydroxyphosphosphoryloxy)-ethylamine (MTP-PE), for example,the submicron oil-in-water emulsion known as “MF59” (InternationalPublication No. WO90/14837; U.S. Pat. Nos. 6,299,884 and 6,451,325, andOtt et al., in Vaccine Design The Subunit and Adjuvant Approach (Powell,M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp.277-296). MF59contains 4-5% w/v squalene (e.g. 4.3%), 0.25-0.5% w/vTWEEN®80 (polyoxyethylene sorbitan monooleate), and 0.5% w/v SPAN®85(sorbitan trioleate) and optionally contains various amounts of MTP-PE,formulated into submicron particles using a microfluidizer such as Model110Y microfluidizer (Microfluidics, Newton, Mass.). For example, MTP-PEmay be present in an amount of about 0-500 μg/dose, more preferably0-250 μg/dose and most preferably, 0-100 μg/dose. As used herein, theterm “MF59-0” refers to the above submicron oil-in-water emulsionlacking MTP-PE, while the term MF59-MTP denotes a formulation thatcontains MTP-PE. For instance, “MF59-100” contains 100 μg MTP-PE perdose, and so on. MF69, another submicron oil-in-water emulsion for useherein, contains 4.3% w/v squalene, 0.25% w/v TWEEN®80 (polvoxvethylenesorbitan monooleate), and 0.75% w/v SPAN®85 (sorbitan trioleate) andoptionally MTP-PE. Yet another submicron oil-in-water emulsion is MF75,also known as SAF, containing 10% squalene, 0.4% TWEEN®80(polyoxyethylene sorbitan monooleate), 5% pluronic-blocked polymer L121,and thr-MDP, also microfluidized into a submicron emulsion. MF75-MTPdenotes an MF75 formulation that includes MTP, such as from 100-400 μgMTP-PE per dose.

Submicron oil-in-water emulsions, methods of making the same andimmunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in WO90/14837 and U.S. Pat. Nos.6,299,884 and 6,451,325.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)may also be used as adjuvants in the invention.

C. Saponin Formulations

Saponin formulations, may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponins isolated from thebark of the Quillaia saponaria Molina tree have been widely studied asadjuvants. Saponins can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs.

Saponin compositions have been purified using High Performance ThinLayer Chromatography (HP-TLC) and Reversed Phase High Performance LiquidChromatography (RP-HPLC). Specific purified fractions using thesetechniques have been identified, including QS7, QS17, QS18, QS21, QH-A,QH-B and QH-C. Preferably, the saponin is QS21. A method of productionof QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulationsmay also comprise a sterol, such as cholesterol (see WO96/33739).

Combinations of saponins and cholesterols can be used to form uniqueparticles called Immunostimulating Complexes (ISCOMs). ISCOMs typicallyalso include a phospholipid such as phosphatidylethanolamine orphosphatidylcholine. Any known saponin can be used in ISCOMs.Preferably, the ISCOM includes one or more of Quil A, QHA and QHC.ISCOMs are further described in EP0109942, WO96/11711 and WO96/33739.Optionally, the ISCOMS may be devoid of (an) additional detergent(s).See WO00/07621.

A review of the development of saponin based adjuvants can be found inBarr, et al., Advanced Drug Delivery Reviews (1998) 32:247-271. See alsoSjolander, et al., Advanced Drug Delivery Reviews (1998) 32:321-338.

D. Virosomes and Virus Like Particles (VLPs)

Virosomes and Virus Like Particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin WO03/024480, WO03/024481, and Niikura et al., Virology (2002)293:273-280; Lenz et al., Journal of Immunology (2001) 5246-5355; Pinto,et al., Journal of Infectious Diseases (2003) 188:327-338; and Gerber etal, Journal of Virology (2001) 75(10):4752-4760. Virosomes are discussedfurther in, for example, Gluck et al., Vaccine (2002) 20:B10-B16.Immunopotentiating reconstituted influenza virosomes (IRIV) are used asthe subunit antigen delivery system in the intranasal trivalentINFLEXAL™ product {Mischler & Metcalfe (2002) Vaccine 20 Suppl 5:B17-23}and the INFLUVAC PLUS™ product.

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as:

(1) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylatedMPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipidA with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such“small particles” of 3dMPL are small enough to be sterile filteredthrough a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPSderivatives include monophosphoryl lipid A mimics, such as aminoalkylglucosaminide phosphate derivatives e.g. RC 529. See Johnson et al.(1999) Bioorg Med Chem Lett 9:2273-2278.

(2) Lipid A Derivatives

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in Meraldi et al.,Vaccine (2003) 21:2485-2491; and Pajak, et al., Vaccine (2003)21:836-842.

(3) Immunostimulatory Oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (asequence containing an unmethylated cytosine followed by guanosine andlinked by a phosphate bond). Bacterial double stranded RNA oroligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpGs can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. Optionally, the guanosine may be replaced with ananalog such as 2′-deoxy-7-deazaguanosine. See Kandimalla, et al.,Nucleic Acids Research (2003) 31(9): 2393-2400; WO02/26757 andWO99/62923 for examples of possible analog substitutions. The adjuvanteffect of CpG oligonucleotides is further discussed in Krieg, NatureMedicine (2003) 9(7): 831-835; McCluskie, et al., FEMS Immunology andMedical Microbiology (2002) 32:179-185; WO98/40100; U.S. Pat. Nos.6,207,646; 6,239,116 and 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT. See Kandimalla, et al., Biochemical Society Transactions (2003)31 (part 3): 654-658. The CpG sequence may be specific for inducing aTh1 immune response, such as a CpG-A ODN, or it may be more specific forinducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs arediscussed in Blackwell, et al., J. Immunol. (2003) 170(8):4061-4068;Krieg, TRENDS in Immunology (2002) 23(2): 64-65 and WO01/95935.Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, Kandimalla, et al., BBRC (2003) 306:948-953; Kandimalla, etal., Biochemical Society Transactions (2003) 31(part 3):664-658; Bhagatet al., BBRC (2003) 300:853-861 and WO03/035836.

(4) ADP-Ribosylating Toxins and Detoxified Derivatives Thereof.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (i.e., E. coli heat labile enterotoxin “LT),cholera (“CT”), or pertussis (“PT”). The use of detoxifiedADP-ribosylating toxins as mucosal adjuvants is described in WO95/17211and as parenteral adjuvants in WO98/42375. Preferably, the adjuvant is adetoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use ofADP-ribosylating toxins and detoxified derivatives thereof, particularlyLT-K63 and LT-R72, as adjuvants can be found in the followingreferences: Beignon et al., Infection and Immunity (2002)70(6):3012-3019; Pizza, et al., Vaccine (2001) 19:2534-2541; Pizza, etal., Int. J. Med. Microbiol. (2000) 290(4-5):455-461; Scharton-Kerstenet al., Infection and Immunity (2000) 68(9):5306-5313; Ryan et al.,Infection and Immunity (1999) 67(12):6270-6280; Partidos et al.,Immunol. Lett. (1999) 67(3):209-216; Peppoloni et al., Vaccines (2003)2(2):285-293; and Pine et al., (2002) J. Control Release (2002)85(1-3):263-270. Numerical reference for amino acid substitutions ispreferably based on the alignments of the A and B subunits ofADP-ribosylating toxins set forth in Domenighini et al., Mol. Microbiol.(1995) 15(6):1165-1167.

F. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres (Singh et al. (2001) J. Cont. Rele. 70:267-276) ormucoadhesives such as cross-linked derivatives of polyacrylic acid,polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose. Chitosan and derivatives thereof may also beused as adjuvants in the invention. See WO99/27960.

G. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable and nontoxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide co glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

H. Liposomes

Examples of liposome formulations suitable for use as adjuvants aredescribed in U.S. Pat. Nos. 6,090,406, 5,916,588, and EP 0 626 169.

I. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters. WO99/52549. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol (WO01/21207) as well as polyoxyethylene alkyl ethers orester surfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol (WO01/21152).

Preferred polyoxyethylene ethers are selected from the following group:polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steorylether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

J. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in Andrianov et al.,“Preparation of hydrogel microspheres by coacervation of aqueouspolyphophazene solutions”, Biomaterials (1998) 19(1-3):109-115 and Payneet al., “Protein Release from Polyphosphazene Matrices”, Adv. Drug.Delivery Review (1998) 31(3):185-196.

K. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP), and Nacetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

L. Imidazoquinoline Compounds.

Examples of imidazoquinoline compounds suitable for use adjuvants in theinvention include Imiquimod and its analogues, described further inStanley, Clin Exp Dermatol (2002) 27(7):571-577; Jones, Curr OpinInvestig Drugs (2003) 4(2):214-218; and U.S. Pat. Nos. 4,689,338,5,389,640, 5,268,376, 4,929,624, 5,266,575, 5,352,784, 5,494,916,5,482,936, 5,346,905, 5,395,937, 5,238,944, and 5,525,612.

M. Thiosemicarbazone Compounds.

Examples of thiosemicarbazone compounds, as well as methods offormulating, manufacturing, and screening for compounds all suitable foruse as adjuvants in the invention include those described in WO04/60308.The thiosemicarbazones are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

N. Tryptanthrin Compounds.

Examples of tryptanthrin compounds, as well as methods of formulating,manufacturing, and screening for compounds all suitable for use asadjuvants in the invention include those described in WO04/64759. Thetryptanthrin compounds are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention:

-   -   (1) a saponin and an oil-in-water emulsion (WO99/11241);    -   (2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g.        3dMPL) (see WO94/00153);    -   (3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g.        3dMPL)+a cholesterol;    -   (4) a saponin (e.g., QS21)+3dMPL+IL 12 (optionally+a sterol)        (WO98/57659);    -   (5) combinations of 3dMPL with, for example, QS21 and/or        oil-in-water emulsions (See European patent applications        0835318, 0735898 and 0761231);    -   (6) SAF, containing 10% squalene, 0.4% TWEEN®80 (polyoxyethylene        sorbitan monooleate), 5% PLURONIC®-block polymer L121, and        thr-MDP, either microfluidized into a submicron emulsion or        vortexed to generate a larger particle size emulsion.    -   (7) RIBI™ adjuvant system (RAS), (Ribi Immunochem) containing 2%        Squalene, 0.2% Tween 80, and one or more bacterial cell wall        components from the group consisting of monophosphorylipid A        (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS),        preferably MPL+CWS (DETOX™); and    -   (8) one or more mineral salts (such as an aluminum salt)+a        non-toxic derivative of LPS (such as 3dPML).    -   (9) one or more mineral salts (such as an aluminum salt)+an        immunostimulatory oligonucleotide (such as a nucleotide sequence        including a CpG motif).

O. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophagecolony stimulating factor, and tumor necrosis factor.

Aluminum salts and MF59 are preferred adjuvants for use with injectableinfluenza vaccines. Bacterial toxins and bioadhesives are preferredadjuvants for use with mucosally-delivered vaccines, such as nasalvaccines.

The contents of all of the above cited patents, patent applications andjournal articles are incorporated by reference as if set forth fullyherein.

Therapeutic Methods

The invention provides the compositions described above for use intherapy, The invention provides the compositions described above forinducing or increasing an immune response to S. pyogenes. The inventionprovides methods for inducing or increasing an immune response to S.pyogenes using the compositions described above. The immune response ispreferably protective and can include antibodies and/or cell-mediatedimmunity (including systemic and mucosal immunity). Immune responsesinclude booster responses.

Teenagers and children, including toddles and infants, can receive avaccine for prophylactic use; therapeutic vaccines typically areadministered to teenagers or adults. A vaccine intended for children mayalso be administered to adults e.g., to assess safety, dosage,immunogenicity, etc.

Diseases caused by Streptococcus pyogenes which can be prevented ortreated according to the invention include, but are not limited to,pharyngitis (such as streptococcal sore throat), scarlet fever,impetigo, erysipelas, cellulitis, septicemia, toxic shock syndrome,necrotizing fasciitis, and sequelae such as rheumatic fever and acuteglomerulonephritis. The compositions may also be effective against otherstreptococcal bacteria, e.g., GBS.

Tests to Determine the Efficacy of the Immune Response

One way of assessing efficacy of therapeutic treatment involvesmonitoring GAS infection after administration of the composition of theinvention. One way of assessing efficacy of prophylactic treatmentinvolves monitoring immune responses against the mutant GAS57 antigensin the compositions of the invention after administration of thecomposition.

Another way of assessing the immunogenicity of the component proteins ofthe immunogenic compositions of the present invention is to mutant GAS57antigens recombinantly and to screen patient sera or mucosal secretionsby immunoblot. A positive reaction between the protein and the patientserum indicates that the patient has previously mounted an immuneresponse to the protein in question; i.e., the protein is an immunogen.This method may also be used to identify immunodominant proteins and/orepitopes.

Another way of checking efficacy of therapeutic treatment involvesmonitoring GAS infection after administration of the compositions of theinvention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses both systemically (such asmonitoring the level of IgG1 and IgG2a production) and mucosally (suchas monitoring the level of IgA production) against GAS57 afteradministration of the composition. Typically, serum specific antibodyresponses are determined post-immunization but pre-challenge whereasmucosal specific antibody body responses are determinedpost-immunization and post-challenge.

The vaccine compositions of the present invention can be evaluated in invitro and in vivo animal models prior to host, e.g., human,administration. Particularly useful mouse models include those in whichintraperitoneal immunization is followed by either intraperitonealchallenge or intranasal challenge.

The efficacy of immunogenic compositions of the invention can also bedetermined in vivo by challenging animal models with GAS, e.g., guineapigs or mice, with the immunogenic compositions. The immunogeniccompositions may or may not be derived from the same serotypes as thechallenge serotypes.

In vivo efficacy models include but are not limited to: (i) a murineinfection model using human GAS serotypes; (ii) a murine disease modelwhich is a murine model using a mouse-adapted GAS strain, such as theM23 strain which is particularly virulent in mice, and (iii) a primatemodel using human GAS isolates.

The immune response may be one or both of a Th1 immune response and aTh2 response. The immune response may be an improved or an enhanced oran altered immune response. The immune response may be one or both of asystemic and a mucosal immune response. Preferably the immune responseis an enhanced system and/or mucosal response.

An enhanced systemic and/or mucosal immunity is reflected in an enhancedTh1 and/or Th2 immune response. Preferably, the enhanced immune responseincludes an increase in the production of IgG1 and/or IgG2a and/or IgA.

Preferably the mucosal immune response is a Th2 immune response.Preferably, the mucosal immune response includes an increase in theproduction of IgA.

Activated Th2 cells enhance antibody production and are therefore ofvalue in responding to extracellular infections. Activated Th2 cells maysecrete one or more of IL-4, IL-5, IL-6, and IL-10. A Th2 immuneresponse may result in the production of IgG1, IgE, IgA and memory Bcells for future protection.

A Th2 immune response may include one or more of an increase in one ormore of the cytokines associated with a Th2 immune response (such asIL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgG1,IgE, IgA and memory B cells. Preferably, the enhanced Th2 immuneresponse will include an increase in IgG1 production.

A Th1 immune response may include one or more of an increase in CTLs, anincrease in one or more of the cytokines associated with a Th1 immuneresponse (such as IL-2, IFNγ, and TNFβ), an increase in activatedmacrophages, an increase in NK activity, or an increase in theproduction of IgG2a. Preferably, the enhanced Th1 immune response willinclude an increase in IgG2a production.

Immunogenic compositions of the invention, in particular, immunogeniccomposition comprising one or more mutant GAS57 antigens of the presentinvention may be used either alone or in combination with other GASantigens optionally with an immunoregulatory agent capable of elicitinga Th1 and/or Th2 response.

The invention also comprises an immunogenic composition comprising oneor more immunoregulatory agent, such as a mineral salt, such as analuminium salt and an oligonucleotide containing a CpG motif. Mostpreferably, the immunogenic composition includes both an aluminium saltand an oligonucleotide containing a CpG motif. Alternatively, theimmunogenic composition includes an ADP ribosylating toxin, such as adetoxified ADP ribosylating toxin and an oligonucleotide containing aCpG motif. Preferably, one or more of the immunoregulatory agentsinclude an adjuvant. The adjuvant may be selected from one or more ofthe group consisting of a Th1 adjuvant and Th2 adjuvant.

The compositions of the invention will preferably elicit both a cellmediated immune response as well as a humoral immune response in orderto effectively address a GAS infection. This immune response willpreferably induce long lasting (e.g., neutralizing) antibodies and acell mediated immunity that can quickly respond upon exposure to one ormore GAS antigens.

In one particularly preferred embodiment, the immunogenic compositioncomprises one or more mutant GAS57 antigen(s) which elicit(s) aneutralizing antibody response and one or more mutant GAS57 antigen(s)which elicit(s) a cell mediated immune response. In this way, theneutralizing antibody response prevents or inhibits an initial GASinfection while the cell-mediated immune response capable of elicitingan enhanced Th1 cellular response prevents further spreading of the GASinfection.

Compositions of the invention will generally be administered directly toa patient. The compositions of the present invention may beadministered, either alone or as part of a composition, via a variety ofdifferent routes. Certain routes may be favored for certaincompositions, as resulting in the generation of a more effective immuneresponse, preferably a CMI response, or as being less likely to induceside effects, or as being easier for administration.

Delivery methods include parenteral injection (e.g., subcutaneous,intraperitoneal, intravenous, intramuscular, or interstitial injection)and rectal, oral (e.g., tablet, spray), vaginal, topical, transdermal(e.g., see WO 99/27961), transcutaneous (e.g., see WO02/074244 andWO02/064162), intranasal (e.g., see WO03/028760), ocular, aural, andpulmonary or other mucosal administration.

By way of example, the compositions of the present invention may beadministered via a systemic route or a mucosal route or a transdermalroute or it may be administered directly into a specific tissue. As usedherein, the term “systemic administration” includes but is not limitedto any parenteral routes of administration. In particular, parenteraladministration includes but is not limited to subcutaneous,intraperitoneal, intravenous, intraarterial, intramuscular, orintrasternal injection, intravenous, intraarterial, or kidney dialyticinfusion techniques. Preferably, the systemic, parenteral administrationis intramuscular injection. As used herein, the term “mucosaladministration” includes but is not limited to oral, intranasal,intravaginal, intrarectal, intratracheal, intestinal and ophthalmicadministration.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunization scheduleand/or in a booster immunization schedule. In a multiple dose schedulethe various doses may be given by the same or different routes e.g., aparenteral prime and mucosal boost, a mucosal prime and parenteralboost, etc.

The compositions of the invention may be prepared in various forms. Forexample, a composition can be prepared as an injectable, either as aliquid solution or a suspension. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g., a lyophilized composition). A composition can beprepared for oral administration, such as a tablet or capsule, as aspray, or as a syrup (optionally flavored). A composition can beprepared for pulmonary administration, e.g., as an inhaler, using a finepowder or a spray. A composition can be prepared as a suppository orpessary. A composition can be prepared for nasal, aural or ocularadministration e.g., as drops. A composition can be in kit form,designed such that a combined composition is reconstituted just prior toadministration to a patient. Such kits may comprise one or more mutantGAS57 or other antigens in liquid form and one or more lyophilizedantigens.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of mutant GAS57 or other antigens (or nucleic acidmolecules encoding the antigens), as well as any other components, asneeded, such as antibiotics. An “immunologically effective amount” is anamount which, when administered to an individual, either in a singledose or as part of a series, increases a measurable immune response orprevents or reduces a clinical symptom.

The immunogenic compositions of the present invention may beadministered in combination with an antibiotic treatment regime. In oneembodiment, the antibiotic is administered prior to administration ofthe antigen of the invention or the composition comprising the one ormore mutant GAS57 antigens of the invention.

In another embodiment, the antibiotic is administered subsequent to theadministration of a mutant GAS57 antigen of the invention. Examples ofantibiotics suitable for use in the treatment of a GAS infection includebut are not limited to penicillin or a derivative thereof orclindamycin, cephalosporins, glycopeptides (e.g., vancomycin), andcycloserine.

The amount of active agent in a composition varies, however, dependingupon the health and physical condition of the individual to be treated,age, the taxonomic group of individual to be treated (e.g., non-humanprimate, primate, etc.), the capacity of the individual's immune systemto synthesize antibodies, the degree of protection desired, theformulation of the vaccine, the treating doctor's assessment of themedical situation, and other relevant factors. The amount will fall in arelatively broad range which can be determined through routine trials.

Kits

The invention also provides kits comprising one or more containers ofcompositions of the invention. Compositions can be in liquid form or canbe lyophilized, as can individual antigens. Suitable containers for thecompositions include, for example, bottles, vials, syringes, and testtubes. Containers can be formed from a variety of materials, includingglass or plastic. A container may have a sterile access port (forexample, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle).

The kit can further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, or dextrose solution. It can also contain othermaterials useful to the end-user, including other buffers, diluents,filters, needles, and syringes. The kit can also comprise a second orthird container with another active agent, for example an antibiotic.

The kit can also comprise a package insert containing writteninstructions for methods of inducing immunity against S. pyogenes or fortreating S. pyogenes infections. The package insert can be an unapproveddraft package insert or can be a package insert approved by the Food andDrug Administration (FDA) or other regulatory body.

All patents, patent applications, and references cited in thisdisclosure are expressly incorporated herein by reference. The abovedisclosure generally describes the present invention. A more completeunderstanding can be obtained by reference to the following specificexamples, which are provided for purposes of illustration only and arenot intended to limit the scope of the invention.

EXAMPLE 1 Purified Wild-Type Recombinant GAS57 Cleaves IL-8 and OtherCXC Cytokines

Wild-type GAS57 was expressed and purified from E. coli either asHis-tagged or untagged protein. All variants were expressed without theN-terminal leader sequence and without the C-terminal transmembranedomain (see SEQ ID NOS:78 and 79). In detail, the gas57 gene was PCRamplified from M1_SF370 genome using the following primers:

(SEQ ID NO: 69) 57F, GTGCGT

GCAGATGAGCTAAGCA (SEQ ID NO: 70) 57R,

(SEQ ID NO: 71) 57stopR,

.

Primers 57F and 57R were used to obtain the His-tagged form, whileprimers 57F and 57stopR were used to obtain the untagged form. The PCRproducts were digested with Ndel-Xhoi and ligated respectively withpET21b+ and pet24b+ cut with the same enzymes.

E. coli BL21(DE3) electrocompetent cells were transformed with theligation reactions. Kanamycin resistant colonies carrying the plasmidwith the correct insert (pET21_(—)57his and pET24_(—)57) were identifiedby colony PCR, and the GAS57 gene was sequenced from one of the positiveclones.

The positive clone expressing GAS57 was grown in liquid culture at 25°C. under agitation, and the expression of the recombinant proteins wasobtained by adding to the culture 1 mM IPTG. Purification of theHis-tagged GAS57 protein was carried out using metal ion affinitychromatography (IMAC). Purification of the untagged form of GAS57 wasaccomplished using three chromatographic steps: ion exchangechromatography (Q SEPHAROSE® HP), hydroxylapatite chromatography and gelfiltration chromatography. FIGS. 7 and 8 show SDS-PAGE analyses ofpurified wild-type proteins.

In order to test GAS57 proteolytic activity, IL-8 was incubated with twodifferent concentrations of purified GAS57 for increasing times and runon an SDS-polyacrylamide gel to demonstrate the conversion of theoriginal 8 kDa IL-8 protein into the cleaved inactive 6 kDa protein. Theresults are shown in FIG. 1.

FIG. 12 shows the results of similar experiments in which variouschemokines (10 μg/ml) were incubated with or without GAS57 (1 μg/ml) at37° C. for 24 hours. Samples were than run on 18% SDS-polyacrylamidegel.

EXAMPLE 2 Preparation Of GAS57 Mutants

By comparison with C5a protease (FIG. 2), three amino acids in the GAS57were identified that putatively constitute the catalytic site of theprotease: D151, H279 and S617. In order to obtain an inactive form ofthe enzyme, nucleotide substitutions resulting in amino acid changesD151A and/or S617A were introduced in the GAS57 coding sequence bySplicing by Overlapping Extension PCR(SOE-PCR).

Substitution D151A

Three PCR reactions were carried out:

PCR reaction Template Primers PCR1 (360 genomic  57F, GTGCGT

GCAGATGAGCTAAGCA; bps) SF370 SEQ ID NO: 69 57mutDR1, CCCTGT

AATAACTGCGAC; SEQ ID NO: 72 PCR2 (910 genomic  57mutDF1, cgCAGTTATT

ACAGGGAT, bp) SF370 SEQ ID NO: 73 57mutSalR, CTGACTGA

AGACTCTGAATAGATG, SEQ ID NO: 74 PCR3 (1270 PCR1,  57F bps) PCR257mutSalR

PCR product 3 was then digested with Nde-Sal and introduced inpET21_(—)57his digested with the same enzymes. Clones containing thecorrect in-frame substitutions (pET21_(—)57his_D151A) were selected byDNA sequencing.

Substitution S617A

Three PCR reactions were carried out:

PCR reaction Template Primers PCR4 (517 genomic 57mutSalF, bp) SF370CTGACTGA

TTTAAAGACATAAAAGATAG; SEQ ID NO: 75 57mutSR1, GAGAGGCCAT

TGTTCCTG; SEQ ID NO: 76 PCR6 (4740 genomic 57mutSF1, GGAACA

ATGGCCTCTCCT; bp) SF370 SEQ ID NO: 77 57R PCR6 (5257 PCR4, 57FmutSalFbp) PCR5 57R

PCR product 6 was then digested with Sal-Xho and introduced inpET21_(—)57his digested with the same enzymes. Clones containing thecorrect in-frame substitutions (pET21_(—)57his_S617A) were selected byDNA sequencing.

Substitution D151A+S617A

PCR product 6 was digested Sal-Xho and introduced inpET21_(—)57his_D151A digested with the same enzymes. Clones containingthe correct in-frame substitutions (pET21_(—)57his_D151A+S617A) wereselected by DNA sequencing.

The single and double mutant proteins were expressed and purified asdescribed above for wild-type GAS57 in Example 1.

EXAMPLE 3 Point Mutation D151A Results in Inactivation of GAS57Proteolytic Activity

GAS57 mutant D151A was expressed as a recombinant His-tagged protein.Two types of assays demonstrated that this mutant has lost the abilityto cleave IL-8.

SDS-PAGE

IL-8 was incubated with wild-type GAS57 or the GAS57 mutant D151A. Theincubation mixtures were loaded on SDS-PAGE and revealed by silverstaining. The results are shown in FIG. 3. Wild-type GAS57 (lanes 2 and3) released two bands: 8 kDa (active form) and 6 kDa (inactive cleavedIL-8). In contrast, the GAS57 D151A mutant released only one band, whichcorresponded to uncleaved IL-8, as in the control reaction (withoutenzyme).

ELISA

IL-8 was incubated with wild-type GAS57 or the GAS57 mutant D151A atthree different concentrations, and the incubation mixtures were testedfor the presence of uncleaved IL-8 using an antibody which is specificfor the cytokine but which is unable to recognize the cleaved inactiveform. The results are shown in FIG. 4, expressed as percentage ofuncleaved IL-8 after 0, 8 and 24 h reactions, and were calculated asfollows:

$\frac{\left\lbrack {{IL}\text{-}8\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{reaction}\mspace{14mu}{mix}} \right\rbrack}{\left\lbrack {{IL}\text{-}8\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{control}\mspace{14mu}{mix}} \right\rbrack,} \times 100$where “control mix” is the reaction mix without the enzyme at time point0.

As shown in FIG. 4, wild-type GAS57 almost completely inactivated IL-8after 8 hours, even at the lower concentration, while no inactivationwas observed for IL-8 treated with the mutant enzyme.

EXAMPLE 4 GAS57 Mutant S617A and GAS57 Double Mutant D151A+S617A do notCleave IL-8

GAS57 mutant S617A and GAS57 double mutant D151A+S617A were expressed asHis-tagged proteins and were tested in IL-8 inactivation experiments asdescribed in Example 2.

SDS-PAGE

IL-8 was incubated with either wild-type GAS57 (His-tagged or tag-less),or each of the GAS57 mutants D151A, S617A and D151AS+S617A for 24 hours.The incubation mixtures were loaded on an SDS-polyacrylamide gel andrevealed by silver staining. The results of two experiments are shown inFIGS. 5A and 5B. Both the GAS57 S617A mutant and the GAS D151+S617Amutant are unable to cleave IL-8, even at a 100-fold higherconcentration than wild-type GAS57.

ELISA

The same samples were used to perform an ELISA assay which confirmedthat the single and double amino acid substitutions eliminate theability of GAS57 to cleave IL-8. The results, which are shown in FIG. 6,demonstrate that the mutants release 100% of uncleaved IL-8 after 24 hincubation, compared to 20-40% released by wild-type GAS57.

EXAMPLE 5 The Protective Capacity of GAS57 Mutants is Similar to thatObtained with Wild-Type GAS57

The GAS57 mutants D151A and D151A+S617A were used to immunize mice totest their capacity to confer protection against GAS lethal challenge incomparison to wild-type GAS57. The results of two experiments (20 miceeach) are summarized below and expressed as average % survival.

NO. MICE NO. DEAD % SURVIVAL PBS + Freund 40 26 35 192 M1 + Freund 20 0100 57 WT + Freund 40 12 70 57 D151A + Freund 40 6 85 57 D151A-S617A +Freund 40 9 78

EXAMPLE 6 Purified Inactive Mutants Appear as a Single Peptide Comparedto Wild-Type GAS57, which Exists Only in the Form of Two Non CovalentlyAssociated Protein Fragments

Wild-type GAS57 is obtained mainly in the form of two fragments, one ofabout 23 kDa and a one of 150 kDa. The two fragments are not separatedin Ni-chelating affinity purification or by gel filtration, but appearas two different bands on SDS-PAGE (FIG. 7). N-terminal sequencingconfirmed that the 23 kDa fragment is the N-terminal portion of GAS57(amino acids 34-244 of SEQ ID NO:1) while the 150 kDa fragment is theC-terminal region (amino acids 245-1603 of SEQ ID NO:1).

In contrast to wild-type GAS57, GAS57 mutants of the invention areobtained as proteins of higher molecular weight (174 kDa), and the 23kDa band is absent (see FIG. 8, which shows the results of an experimentin which partially purified wild-type GAS57 and GAS57 mutants wereloaded on SDS-polyacrylamide gels).

EXAMPLE 7 Dose-Dependent Inhibition of GAS57-Mediated IL-8 Cleavage byPolyclonal Antisera

Mouse antisera specific for GAS57, wild type and inactive mutants, wereproduced by immunizing CD1 mice with the purified recombinant proteins.

IL-8 (10 μg/ml) was incubated with wild-type GAS57 with or without GAS57antiserum (1:50 and 1:5000) in two different conditions: (1) 8 hourincubation, 0.1 μg/ml of GAS57 and (2) 24 hour incubation, 0.05 μg/ml ofGAS57. The incubation mixtures were then tested for the presence ofuncleaved IL-8 by ELISA. The results shown in FIGS. 9A and 9Bdemonstrated a dose-dependent inhibition of GAS57-mediated IL-8 cleavageby the mouse antiserum.

The invention claimed is:
 1. A method of raising an immune responseagainst Streptococcus pyogenes comprising administering to a patient animmunologically effective amount of a composition comprising: (a) amutant Spy0416 antigen comprising the amino acid sequence SEQ ID NO:1but for an amino acid substitution at one or more amino acid positionsselected from the group consisting of amino acids D151, H279, and S617;and (b) a pharmaceutically acceptable carrier.
 2. The method of claim 1,wherein the mutant Spy0416 antigen comprises the amino acidsubstitutions at the amino acids D151 and S617.
 3. The method of claim1, wherein the mutant Spy0416 antigen comprises the amino acidsubstitution at the amino acid D151.
 4. The method of claim 1, whereinthe mutant Spy0416 antigen comprises the amino acid substitution at theamino acid H279.
 5. The method of claim 1, wherein the mutant Spy0416antigen comprises the amino acid substitution at the amino acid S617. 6.The method of claim 1, wherein the composition further comprises anadjuvant.
 7. The method of claim 6, wherein the adjuvant comprises analuminium salt.
 8. The method of claim 6, wherein the adjuvant comprisesa sub-micron oil in water emulsion comprising squalene, polyoxyethylenesorbitan monooleate, and sorbitan trioleate.
 9. The method of claim 2,wherein the composition further comprises an adjuvant.
 10. The method ofclaim 9, wherein the adjuvant comprises an aluminium salt.
 11. Themethod of claim 9, wherein the adjuvant comprises a sub-micron oil inwater emulsion comprising squalene, polyoxyethylene sorbitan monooleate,and sorbitan trioleate.
 12. The method of claim 3, wherein thecomposition further comprises an adjuvant.
 13. The method of claim 12,wherein the adjuvant comprises an aluminium salt.
 14. The method ofclaim 12, wherein the adjuvant comprises a sub-micron oil in wateremulsion comprising squalene, polyoxyethylene sorbitan monooleate, andsorbitan trioleate.
 15. The method of claim 4, wherein the compositionfurther comprises an adjuvant.
 16. The method of claim 15, wherein theadjuvant comprises an aluminium salt.
 17. The method of claim 15,wherein the adjuvant comprises a sub-micron oil in water emulsioncomprising squalene, polyoxyethylene sorbitan monooleate, and sorbitantrioleate.
 18. The method of claim 5, wherein the composition furthercomprises an adjuvant.
 19. The method of claim 18, wherein the adjuvantcomprises an aluminium salt.
 20. The method of claim 18, wherein theadjuvant comprises a sub-micron oil in water emulsion comprisingsqualene, polyoxyethylene sorbitan monooleate, and sorbitan trioleate.